Methods of treatment and pharmaceutical compositions using bcn057 or bcn512

ABSTRACT

The present disclosure is directed to method of treatment for treating or ameliorating various conditions pertaining such as bone marrow recovery (or blood cell production), fibrosis, inflammatory diseases, inhibition of cancer cell growth, propagation or malignancy, thrombocytopenia, wound healing, and conditions related to stem cells by the administration of BCN057, 512, or an analog thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patent Ser. No.16/292,266, filed Mar. 4, 2019, which is a Continuation of applicationSer. No. 15/764,162 filed Oct. 15, 2017 which claims priority tonon-provisional application U.S. Ser. No. 15/784,162, filed Oct. 15,2017; and provisional application U.S. Ser. No. 62/408,809, filed Oct.16, 2016, herein incorporated by reference in their entirety. Thisapplication is also related to U.S. Ser. No. 13/813,923 and U.S. Ser.No. 14/889,719, herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The discovery of compounds that are capable of mitigating the process ofnormal tissue damage from radiation during radiotherapy, accidents, oracts of war is of great importance. Most currently available treatmentsfor radiation exposure are free radical scavengers that reduce initialradiation-induced DNA damage and work best if added just before or atthe time of irradiation. Because of this, these compounds are notpractical countermeasures for a radiation incident. In that case, thesearch for radiomitigators—agents with robust, prolonged efficacy, broadspecificity, and minimal toxicity is of great importance. In addition,for applications such as radio-therapy for cancer, the compound shouldprotect the normal tissue, but not the cancerous tissue.

The present application provides new uses for molecules that wereoriginally identified to protect normal cells from radiation inducedcell death. Fundamental to radiation exposure and injury are DNA strandbreaks, resulting in genetic instability and DNA deletions which areinvolved in cell death, cellular dysfunction, as well as longer termconsequences such as birth defects and cancer.

The compounds disclosed herein were first described in U.S. Ser. No.13/813,923 and U.S. Ser. No. 14/889,719. The present invention providesnew methods of use for these compounds, in particular the compoundsBCN057 (also called YEL002) and 512, also called BCN512.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of increasinghematopoiesis in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount ofBCN057, BCN512, or an analog thereof.

In one embodiment, the subject has leukemia, AML, ALL, bone marrowablation, bone marrow transplant, bone marrow suppression due toradiation or chemotherapy, a platelet disorder, or clinical radiationrelated exposure.

In one embodiment, the platelet disorder is caused by bone marrowfailure (e.g., aplastic anemia, paroxysmal nocturnal hemoglobinuria,Shwachman-Diamond syndrome), bone marrow suppression (e.g., frommedication, chemotherapy, or irradiation as discussed above), chronicalcohol abuse, congenital macrothrombocytopenias (e.g., Alport syndrome,Bernard-Soulier syndrome, Fanconi anemia, platelet-type or pseudo-vonWillebrand disease, Wiskott-Aldrich syndrome), infection (e.g.,cytomegalovirus, Epstein-Barr virus, hepatitis C virus, HIV, mumps,parvovirus B19, rickettsia, rubella, varicella-zoster virus),myelodysplastic syndrome, neoplastic marrow infiltration, or nutritionaldeficiencies (vitamin B12 and folate).

In one aspect, the invention provides a method of inhibiting cancer cellgrowth in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of BCN057, BCN512 oran analog thereof, wherein the cancer is selected from the groupconsisting of renal cancer, prostate cancer, non-small cell lung cancer,head and neck cancers, breast cancer, colon cancer, ovarian, leukemia,skin cancer such as melanoma, central nervous system cancers includingpediatric brain cancers and adult brain cancers.

In one aspect, the invention provides a method of preventing lateeffects of clinical radiation, the method comprising administering tothe subject a therapeutically effective amount of BCN057, BCN512 or ananalog thereof, wherein the effects are reduction of tissue fibrosis,reduction in hormonal deficits, reduction in neurological impairmentfrom radiation, reduction in growth retardation from radiationtreatment, reduction of pulmonary, prostate, colon or kidney damage fromradiation, reduction in leukemia arising from radiation treatment

In one aspect, the invention provides a method of treating fibrosis in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of BCN057, BCN512, or ananalog thereof.

In one embodiment, the fibrosis is a fibrotic disease selected from thegroup consisting of idiopathic pulmonary fibrosis, liver fibrosis,gastrointestinal fibrosis and renal fibrosis from kidney dialysis

In one embodiment, the fibrotic disease is pulmonary fibrosis,idiopathic pulmonary fibrosis, acute respiratory distress syndrome,cystic fibrosis, non-cystic fibrosis bronchiectasis, cirrhosis, liverfibrosis (caused, for example by chronic viral hepatitis B or C),endomyocardial fibrosis, old myocardial infarction, atrial fibrosis,mediastinal fibrosis (soft tissue of the mediastinum), myelofibrosis(bone marrow), retroperitoneal fibrosis, progressive massive fibrosis,nephrogenic systemic fibrosis, Crohn's disease, gastrointestinalfibrosis, keloid conditions, scleroderma/systemic sclerosis,arthofibrosis, peyronie's disease, dupuytren's contracture, oralsubmucous fibrosis, or adhesive capsulitis.

In one aspect, the invention provides a method of improving wound andtissue healing in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount ofBCN057, BCN512 or an analog thereof.

In one embodiment, the wound is a dermal wound. In one embodiment, thewound is caused by sun, radiation or heat exposure.

In one embodiment, the analog is selected from the group consisting ofFormula IB-H. In one embodiment, the compound is Formula IA. In oneembodiment, the analog is selected from the group consisting of FormulaIIB-H. In one embodiment, the compound is Formula IIA.

In one embodiment, the subject received radiation therapy.

In one aspect, the invention provides a pharmaceutical composition ofBCN057 comprising 100 mM methanesulfonic acid/10% povidone (PVP); 100 mMMSA/2% benzyl alcohol/2% N-methylpyrrolidone (NMP).

In one embodiment, the composition comprises 30 wt % Captisol(SBE-beta-CD) and 100 mM MSA. In another embodiment, the compositionfurther comprises 30 wt % Captisol (SBE-beta-CD) and 100 mM MSA at pH4.0 or lower.

In one aspect, the invention provides a nanoparticle pharmaceuticalcomposition of BCN512.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Inhibition of RPMI-8226, K-562, and CCRF-CEM cell lines byBCN057.

FIG. 2: Inhibition of breast cancer cell lines: T-470, MDA-MB-231/ATCC,H5578T, BT-549, and MDA-MB-468; the CNS cancer lines: SNB-19, SNB-75,SF-539; Colon cancer cell line HCT-116; Melanoma cell lines: UACC-62SK-MEL-5; non-small cell lung cancer cell lines: HOP-92, EKVX, NCI-H23;ovarian cancer cell lines IGROV1, NCI/ADR-RES, OVCAR-4; prostate cancercell line PC-3; and renal cancer cell lines: A498, ACHN, UO-31, CAKI-1,and 786Q by BCN057.

FIG. 3 is a graph of the amount of blood platelets from plasma (mouse)demonstrating through multiple doses and multiple routes of entry thatthe drug stimulates platelet production rapidly.

FIG. 4: Cytokine panel for mice treated with BCN057.

FIG. 5 shows the cytokine analysis of plasma from mice treated withnothing (marked as 0G above), 7 Grey radiation (7G) and 7G+BCN057).

FIG. 6A: Pharmacokinetic profile for C57BL/6 male mice receiving a 10mg/kg sc injection of 512 in a low % deoxycholate formulation. Cmaxobserved is 132.5 ng/ml with a Tmax observed at 2 hours.

FIG. 6B: BCN512 did not increase the number of tumor colonies

FIG. 7A-D: Tumor metastasis models of the number of metastatic tumornodules in C3H, C57B16, and nude mice. (A) weight (C57) over time. (B).Number of LLC nodules (C57) and percentage of LLC nodules. (C) weight(C3H) over time. (D). Number of LLC nodules (C3H) and percentage of LLCnodules.

FIG. 8A-C: (A) Kaplan Meier Plot of Male C3H/HeN mice 7.75Gy 30 Day TBI(n=30) 50 mg/kg BCN057. (B). Kaplan Meier Plot of Female C3H/HeN mice7.75Gy 30 Day TBI (n=10) 25 mg/kg BCN057. (C) Cytokine Profile in PlasmaMale C3H/HeN mice 7.75Gy TBI Day 8 receiving BCN057.

FIG. 9: BCN 512 prevents hematopoietic suppression from total bodyirradiation in mice.

FIG. 10: Bone marrow removed from mice treated with nothing (control) or512, second from left.

FIG. 11A-C: (A) Cancer Cell Proliferation in the presence of BCN057 10mM expressed as a percent of control which is considered 100%. Cancercell proliferation rate (various lines) vs control of 10 ul BCN057 vscontrol which is DMSO alone. (B)-(C) Panel name, cell name and % ofcontrol.

FIG. 12: BCN 512 treatment induced luciferase activity, indicatingactivation of canonical Wnt signaling.

FIG. 13A-C: BCN-512 mitigates radiation-induced damage in lungorganoids. Please note that in the untreated group, the organoidstructure was completely lost within 72-96 h of radiation exposure.

FIG. 14A-B: Lgr5 and Fzd5 receptors were co-localized in cells at theBADJ region. Lung epithelial sections from Lgr5-GFP-Cre-ERT mice werestained with chicken anti GFP antibodies (primary antibody; 1:200dilution) and donkey anti-chicken 488 (secondary antibody; 1:200dilution) to detect Lgr5 expression. To detect Fzd5 expression, sectionswere stained with rabbit anti-Fzd (primary antibody; 1:50 dilution) anddonkey anti-rabbit 546 (secondary antibody; 1:200 dilution).Fzd5-positive cells are red and Lgr5-positive cells are green. Cellsco-expressing both receptors are yellow in color (red+green) and areprimarily located at the BADJ region, as indicated with an arrow.

FIG. 15: Number of fibroblasts recovered from co-cultures with 25-foldmore non-irradiated or 2 Gr-irradiated macrophages treated with diluent,BCN 512, LPS, or LPS+BCN 512 after 3 days culture. The blue line is theno macrophage control.

FIG. 16: Flow cytometry profiles of CFSE-labeled fibroblasts co-cultureswith macrophages at a ratio of 25:1 for 3 days in the presence ofdiluent, BCN 512, LPS, or LPS+512. The fluorescence intensity decreasedas the fibroblasts proliferated. Non-activated control macrophagesactually stimulate fibroblast proliferation; the blue line represents nomacrophage control. In contrast, activation with LPS caused theequivalent of a 1-day growth arrest and decreased viability.

FIG. 17: The ratio of viable fibroblasts in irradiated (6 Gr) andnon-irradiated macrophage co-cultures on day 3. Irradiation increasedthe ability of macrophages to support fibroblasts, and 512 decreasedthis effect of irradiation.

FIG. 18A-B: Histologic examination of rodents undergoing local thoracicradiation and lung damage.

FIG. 19A-C: Histologic examination of rodents undergoing local thoracicradiation and lung damage.

FIG. 20: Radiation sites for wound healing experiments.

FIG. 21: Average Draize Scoring. Plot of difference between Erythemadevelopment (blue) (left 6 columns) and Edema (right 6 columns).Radiation treatment alone is blue and BCN057 treatment is red (rightcolumn pair).

FIG. 22: Species 1-4 for BCN057 formulation experiments.

FIG. 23: BCN 512 induces the WINT target gene expression.

FIG. 24A-F: Photos of dermal wound healing from radiation. A-B arecontrol sites where only vehicle and no drug was used subsequent toradiation. C-D are drug treatment sites irradiated identical to thecontrol site but with vehicle containing BCN512 at 10 mg/ml. E-F aredrug treatment sites irradiated identical to the control site but withvehicle containing BCN057 at 10 mg/ml. Photographs of these subjectwounds are also analyzed for Draize scoring in FIGS. 20 and 21.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The compounds and compositions disclosed herein, including BCN512,BCN057 and analogs thereof can be used for treating or amelioratingvarious conditions described herein, such as bone marrow recovery andhematopoiesis, inhibition of cancer cell growth, late effects ofradiation including fibrosis, and wound healing.

Compositions

The structure of compound YEL002/BCN057 is shown below as Formula IA:

Yel002 or BCN057 is also known as3-(3((furan-2-ylmethyl)ammo)-6-methylimidazo[1,2-a]pyridine-2-yl)-6-methoxyquinolin-2(1H)-one.

In some embodiments, the compound is an analog of Formula IA selectedfrom Formulae IB-IH:

A compound of Formula IA, or an analog thereof disclosed herein can beprepared according to established methodology in the art of organicsynthesis. General methods of synthesizing the compound can be found in,e.g., Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: TheDisconnection Approach, second Edition, Wiley, 2010. Exemplary methodsof making the compound is provided in U.S. Ser. No. 13/813,923 and U.S.Ser. No. 14/889,719, herein incorporated by reference in their entirety.The compound also includes a pharmaceutically acceptable salt thereof, aprodrug thereof, a hydrate thereof, a solvate thereof, or a polymorphiccrystal thereof. The compound may be administered as a pharmaceuticalcomposition.

The structure of compound BCN512 is shown below as Formula IIA:

Compound 512 is also known as 1-[(4-nitrobenezene)sulfonyl]-4-phenylpiperazine. Analogs of 512 include compounds of Formula IIB:

wherein:Y¹ and Y² taken together with X form:

and wherein:

X is N; G is N;

Z is absent or selected from substituted or unsubstituted alkyl,heteroalkyl, alkenyl, or alkynyl;R⁴ is absent or selected from substituted or unsubstituted aryl; andR⁵ and R⁶ are each independently absent or lower alkyl.

In one embodiment, the analog is selected from Formulae IIC-E:

A compound of Formula IIA, or an analog thereof disclosed herein, can beprepared according to established methodology in the art of organicsynthesis. General methods of synthesizing the compound can be found in,e.g., Stuart Warren and Paul Wyatt, Workbook for Organic Synthesis: TheDisconnection Approach, second Edition, Wiley, 2010. Exemplary methodsof making the compound is provided in U.S. Ser. No. 13/813,923 and U.S.Ser. No. 14/889,719, herein incorporated by reference in their entirety.The compound also includes a pharmaceutically acceptable salt thereof, aprodrug thereof, a hydrate thereof, a solvate thereof, or a polymorphiccrystal thereof. The compound may be administered as a pharmaceuticalcomposition.

Methods of Treatment

The compounds and compositions disclosed herein can be used for treatingor ameliorating various conditions such as bone marrow recovery andhematopoiesis, inhibition of cancer cell growth, late effects ofradiation including fibrosis, and wound healing.

Bone Marrow Recovery and Hematopoiesis

Some cancer treatments, including radiation therapy and chemotherapy,can affect the bone marrow so that it does not make normal numbers ofblood cells. Similarly, bone marrow transplant may also reduce theproduction of normal numbers of blood cells. When the bone marrowdoesn't work properly, blood cells are not replaced as they normallywould be and blood cell counts drop. Blood cell counts usually start todrop 7-10 days or longer after treatment. The time frame depends on thetype of treatment given.

The lowest level that blood cell counts reach is called the nadir. Eachtype of blood cell has a different nadir and nadir occurs at differenttimes. Because RBCs live the longest, it takes them longer to reachtheir nadir. WBCs and platelets reach their lowest levels about 7-14days after treatment. Symptoms of low blood cell counts are often worseat nadir. Blood cell counts may begin to recover and rise on their own,usually 2-4 weeks after radiation, chemotherapy or transplant treatment,however they may be suppressed for a significant period beyond thepredicating treatment. The present methods speed bone marrow recovery.

Radiation therapy can damage tissues in the bone marrow that make bloodcells. This is especially true if areas that contain large amounts ofbone marrow, such as the pelvic bones, are treated with radiation. Thepresent compositions may be administered prophylactically before theinitiation of radiation therapy, concurrently with radiation therapy, inbetween intervals of radiation therapy, or after radiation therapy.

Bone marrow transplant may lead to suppressed bone marrow recovery.Chemotherapy can also damage tissues in the bone marrow that make bloodcells. For instance, bone marrow toxicity may occur withchemotherapeutic agents such as methotrexate, doxorubicin,cyclophosphamide, ifosfamide, 5-fluorocil, 6-mercaptopurine, cytarabine,gemcitabine, fludarabine, etoposide, bleomycin, irinotecan, topotecan,vinblastine, vincristine, paclitaxel or docetaxel, cisplatin,carboplatin, oxaliplatin and iomustine. In many of these dose regimens,it is a late complication of treatment. In some cases, occurring inpatients having taken a given drug for over a year, and may not beappropriately monitored. People with kidney or liver problems have ahigher risk of bone marrow suppression while on chemotherapy becausetheir bodies can't break down the chemotherapy drugs.

Thus, the present therapies may be administered to patients who areconcurrently prescribed with chemotherapy or after the onset of bonemarrow suppression resulting from the chemotherapy. Furthermore, thepresent compositions may be administered in patients with an extendedperiod of treatment with the expectation that the present therapies willprevent, delay, or attenuate the toxicity towards bone marrow, and speedbone marrow recovery. The present methods could be applied after it isapparent that bone marrow suppression has occurred.

Advanced HIV invention may also lead to bone marrow suppression. Thus,in one embodiment, the present compositions may be administered topatients having HIV, before or after the bone marrow suppression becomesevident.

Symptoms of bone marrow suppression can vary depending on their causeand other factors. It can also vary with the type of blood cell that isaffected.

Anemia (a lower number of red blood cells) causes fatigue, pale skin,lips or nail beds, increased heart rate, tiring easily with exertion,dizziness, shortness of breath, headache, irritability—more often seenin young children. Neutropenia or leukopenia (lower number of whiteblood cells) causes: a greater risk of infection, fever and chills if aninfection is present. Thrombocytopenia (a lower number of platelets)causes easy bruising, bleeding from the nose, gums or mouth, tiny redspots on the skin, or petechiae, blood in the urine, dark or black bowelmovements.

Pancytopenia refers to low levels of all 3 types of blood cells. Thesymptoms could include any or all of the above symptoms.

Furthermore, protection may also occur for other cells in the bonemarrow such as progenitor cells, myeloid cells, stem cells, fibroblast,endo and epithelial cells, and immune cells. Accordingly, in oneembodiment, the administration of the present compounds increases theproduction of or prevents the destruction of progenitor cells, myeloidcells, stem cells, fibroblasts, endo and epithelial cells, and immunecells.

In one embodiment, the present compounds may also be used to spur stemcell self-renewal. Hematopoietic Stem Cells (HSCs) possess the abilityof both multi-potency and self-renewal. Multi-potency is the ability todifferentiate into all functional blood cells. Self-renewal is theability to give rise to HSC itself without differentiation. Since matureblood cells are predominantly short lived, HSC continuously provide moredifferentiated progenitors while properly maintaining the HSC pool sizeproperly throughout life by precisely balancing self-renewal anddifferentiation.

In a further aspect of stem-cell self-renewal, the present compounds maybe used for the stimulation of engrafted stem cells.

In certain embodiments, the therapeutic compounds disclosed hereinincrease production of suppressed cell populations by bone marrow by,e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% or at least 95%. In certain embodiments,the therapeutic compounds disclosed herein increase production ofsuppressed cell populations by bone marrow by, e.g., about 5% to about100%, about 10% to about 100%, about 20% to about 100%, about 30% toabout 100%, about 40% to about 100%, about 50% to about 100%, about 60%to about 100%, about 70% to about 100%, about 80% to about 100%, about10% to about 90%, about 20% to about 90%, about 30% to about 90%, about40% to about 90%, about 50% to about 90%, about 60% to about 90%, about70% to about 90%, about 10% to about 80%, about 20% to about 80%, about30% to about 80%, about 40% to about 80%, about 50% to about 80%, orabout 60% to about 80%, about 10% to about 70%, about 20% to about 70%,about 30% to about 70%, about 40% to about 70%, or about 50% to about70%.

In certain embodiments, the therapeutic compound disclosed hereinincreases production of suppressed cell populations by bone marrow bye.g., at least at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% or at least 95% as compared to apatient not receiving the same treatment. In certain embodiments, thetherapeutic compound disclosed increases production of suppressed cellpopulations by bone marrow by, e.g., about 5% to about 100%, about 10%to about 100%, about 20% to about 100%, about 30% to about 100%, about40% to about 100%, about 50% to about 100%, about 60% to about 100%,about 70% to about 100%, about 80% to about 100%, about 10% to about90%, about 20% to about 90%, about 30% to about 90%, about 40% to about90%, about 50% to about 90%, about 60% to about 90%, about 70% to about90%, about 10% to about 80%, about 20% to about 80%, about 30% to about80%, about 40% to about 80%, about 50% to about 80%, or about 60% toabout 80%, about 10% to about 70%, about 20% to about 70%, about 30% toabout 70%, about 40% to about 70%, or about 50% to about 70% as comparedto a patient not receiving the same treatment.

In certain embodiments, the therapeutic compound disclosed hereinreduces the duration of the symptoms of bone marrow suppression by atleast 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, or 2 years as compared to a patient not receiving thesame treatment.

Thrombocytopenia

Thrombocytopenia is a clinically decreased number of platelets, and maybe independent of bone marrow suppression. It may be caused by adecreased platelet production, increased platelet destruction,sequestration of platelets, or pseudotrombocytopenia. Multiple genetic,immune, or environmental conditions may lead to thrombocytopenia.Decreased platelet production may be caused for example, by bone marrowfailure (e.g., aplastic anemia, paroxysmal nocturnal hemoglobinuria,Shwachman-Diamond syndrome), Bone marrow suppression (e.g., frommedication, chemotherapy, or irradiation as discussed above), Chronicalcohol abuse, Congenital macrothrombocytopenias (e.g., Alport syndrome,Bernard-Soulier syndrome, Fanconi anemia, platelet-type or pseudo-vonWillebrand disease, Wiskott-Aldrich syndrome), Infection (e.g.,cytomegalovirus, Epstein-Barr virus, hepatitis C virus, HIV, mumps,parvovirus B19, rickettsia, rubella, varicella-zoster virus),Myelodysplastic syndrome, Neoplastic marrow infiltration, or Nutritionaldeficiencies (vitamin B12 and folate).

Increased platelet consumption/destruction may be caused by: Alloimmunedestruction (e.g., posttransfusion, neonatal, posttransplantation),Autoimmune syndromes (e.g., antiphospholipid syndrome, systemic lupuserythematosus, sarcoidosis), Disseminated intravascularcoagulation/severe sepsis, Drug-induced thrombocytopenia (caused, forexample, by quinidine, quinine, sulfa-containing antibiotics,interferon, anticonvulsants and gold salts), Heparin-inducedthrombocytopenia, Immune thrombocytopenic purpura*, Infection (e.g.,cytomegalovirus, Epstein-Barr virus, hepatitis C virus, HIV, mumps,parvovirus B19, rickettsia, rubella, varicella-zoster virus,bacteremia), Mechanical destruction (e.g., aortic valve, mechanicalvalve, extracorporeal bypass), Preeclampsia/HELLP syndrome, Thromboticthrombocytopenic purpura (small blood clots suddenly form throughout thebody, using up large numbers of platelets), hemolytic uremic syndrome(which causes sharp drop in platelets, destruction of red blood cellsand impairment of kidney function. Sometimes it can occur in associationwith a bacterial Escherichia coli infection, such as may be acquiredfrom eating raw or undercooked meat), idopathic thrombocytopenic purpura(ITP) (where the body's immune system mistakenly identifies platelets asa threat and forms antibodies that attack them).

Sequestration of platelets leading to thrombocytopenia may be caused by,for example, Chronic alcohol abuse, Dilutional thrombocytopenia (e.g.,hemorrhage, excessive crystalloid infusion), Gestationalthrombocytopenia, Hypersplenism (e.g., distributional thrombocytopenia),Liver disease (e.g., cirrhosis, fibrosis, portal hypertension),Pseudothrombocytopenia, Pulmonary emboli, or Pulmonary hypertension.

In addition, some causes may lead to one or more reasons for a shortageof platelets. For instance, certain infections lead to both a decreasedproduction of platelets as well as an increased destruction ofplatelets. Pregnancy in general may also cause a decrease in plateletcount.

The present compositions can treat thrombocytopenia by increasing theproduction of platelets. Thus, in one embodiment, the presentcompositions may be administered to treat or mitigate thrombocytopeniacaused by conditions which decrease the number of platelets produced. Inanother aspect, the present compositions are administered to treat ormitigate thrombocytopenia caused by conditions which obtain abnormallylow platelet counts by destroying platelets.

In one embodiment, the present compounds increase the platelet count by,e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% or at least 95%.

In certain embodiments, the therapeutic compound disclosed hereinincreases platelet count by e.g., at least at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95% as compared to a patient not receiving the same treatment. Incertain embodiments, the therapeutic compound disclosed herein increasesplatelet count by, e.g., about 5% to about 100%, about 10% to about100%, about 20% to about 100%, about 30% to about 100%, about 40% toabout 100%, about 50% to about 100%, about 60% to about 100%, about 70%to about 100%, about 80% to about 100%, about 10% to about 90%, about20% to about 90%, about 30% to about 90%, about 40% to about 90%, about50% to about 90%, about 60% to about 90%, about 70% to about 90%, about10% to about 80%, about 20% to about 80%, about 30% to about 80%, about40% to about 80%, about 50% to about 80%, or about 60% to about 80%,about 10% to about 70%, about 20% to about 70%, about 30% to about 70%,about 40% to about 70%, or about 50% to about 70% as compared to apatient not receiving the same treatment.

Inflammatory Diseases

Compounds of the current invention may also be used for the treatment orprevention of inflammation and inflammatory diseases. Symptoms ofinflammation include: decreased levels of one or more signaling agentssuch as IL-6 (direct signaling for regenerative activity), IL-10,Epinephrine, IL-4, IL-10, IL-13, IL-1RA, Leukotriene B4-receptorantagonism, LPS binding protein, Soluble recombinant CD-14, SolubleTNF-α receptors, Transforming growth factor-0, and/or Type II IL-1R;Increased levels of one or more signaling agents such as IL1-beta, IL-2,IL-6 (resulting from trans-signaling), IL-8, IL-15, IFN-gamma, Leukemiainhibitory factor, Monocyte chemoattractant protein-1, Monocytechemoattractant protein-2, Neopterin, Neutrophil elastase, PhospholipaseA2, Plasminogen activator inhibitor-1, Platelet activating factor,Prostacyclins, Prostaglandins, Protein kinase, Soluble adhesionmolecules, Thromboxane, TNF-α, Tyrosine kinase, Vasoactiveneuropeptides, TNF-alpha, GCSF, GMCSF, and/or EGF; swelling, cellularinfiltrate (e.g., with lymphocytes and macrophages), and increasedcellular matrix.

Examples of inflammatory conditions, which may be treated or preventedby the administration of a compound of the invention include, but arenot limited to, inflammation of the lungs, joints, connective tissue,eyes, nose, bowel, kidney, liver, skin, central nervous system, vascularsystem and heart. In certain embodiments, inflammatory conditions whichmay be treated by the current invention include inflammation due to theinfiltration of leukocytes or other immune effector cells into affectedtissue. Other relevant examples of inflammatory conditions which may betreated by the present invention include inflammation caused byinfectious agents, including, but not limited to, viruses, bacteriafungi and parasites.

Inflammatory lung conditions include, but are not limited to, asthma,adult respiratory distress syndrome, bronchitis, pulmonary inflammation,pulmonary fibrosis, and cystic fibrosis (which may additionally oralternatively involve the gastro-intestinal tract or other tissue(s)).Inflammatory joint conditions include rheumatoid arthritis, rheumatoidspondylitis, juvenile rheumatoid arthritis, osteoarthritis, goutyarthritis and other arthritic conditions. Eye diseases with aninflammatory component include, but are not limited to, uveitis(including iritis), conjunctivitis, scleritis, keratoconjunctivitissicca, and retinal diseases, including, but not limited to, diabeticretinopathy, retinopathy of prematurity, retinitis pigmentosa, and dryand wet age-related macular degeneration. Inflammatory bowel conditionsinclude chronic inflammation of all or part of the digestive tract,Crohn's disease (including general inflammation of the digestive tract,inflammation of the bowel wall, inflammation of the ileum, inflammationof the colon, and the sequelae of Chrohn's disease, such asfibrostenosis, fistula and obstructions), ulcerative colitis (includingulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis,and acute severe ulcerative colitis), and distal proctitis. In oneaspect, the inflammatory bowel disease also includes collagenouscolitis, lymphocytic colitis. In one embodiment, the inflammatory bowelconditions are Crohn's disease, ulcerative colitis, and distalproctitis. General symptoms of these conditions include: diarrhea,abdominal pain, cramping, fatigue, anal pain, blood in the stool,reduced appetite, and unintended weight loss.

Inflammatory skin diseases include, but are not limited to, conditionsassociated with cell proliferation, such as psoriasis, eczema anddermatitis, (e.g., eczematous dermatitides, topic and seborrheicdermatitis, allergic or irritant contact dermatitis, eczema craquelee,photoallergic dermatitis, phototoxic dermatitis, phytophotodermatitis,radiation dermatitis, and stasis dermatitis). Other inflammatory skindiseases include, but are not limited to, scleroderma, ulcers anderosions resulting from trauma, burns, bullous disorders, or ischemia ofthe skin or mucous membranes, several forms of ichthyoses, epidermolysisbullosae, hypertrophic scars, keloids, cutaneous changes of intrinsicaging, photoaging, frictional blistering caused by mechanical shearingof the skin and cutaneous atrophy resulting from the topical use ofcorticosteroids. Additional inflammatory skin conditions includeinflammation of mucous membranes, such as cheilitis, chapped lips, nasalirritation, mucositis and vulvovaginitis.

Inflammatory disorders of the endocrine system include, but are notlimited to, autoimmune thyroiditis (Hashimoto's disease), Type Idiabetes, and acute and chronic inflammation of the adrenal cortex.Inflammatory conditions of the cardiovascular system include, but arenot limited to, coronary infarct damage, peripheral vascular disease,myocarditis, vasculitis, revascularization of stenosis,artherosclerosis, and vascular disease associated with Type II diabetes.

Inflammatory condition of the kidney include, but are not limited to,glomerulonephritis, interstitial nephritis, lupus nephritis, nephritissecondary to Wegener's disease, acute renal failure secondary to acutenephritis, Goodpasture's syndrome, post-obstructive syndrome, tubularischemia, irritable bowel disorder, or inflammation induced colonmalignancies.

Inflammatory conditions of the liver include, but are not limited to,hepatitis (arising from viral infection, autoimmune responses, drugtreatments, toxins, environmental agents, or as a secondary consequenceof a primary disorder), biliary atresia, primary biliary cirrhosis andprimary sclerosing cholangitis.

Inflammatory conditions of the central nervous system include, but arenot limited to, multiple sclerosis and neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, or dementia associated withHIV infection.

Other inflammatory conditions include periodontal disease, tissuenecrosis in chronic inflammation, endotoxin shock, smooth muscleproliferation disorders, graft versus host disease, tissue damagefollowing ischemia reperfusion injury, idiopathic pulmonary fibrosis,and tissue rejection following transplant surgery.

In one embodiment, the condition is idiopathic pulmonary fibrosis.Symptoms of idiopathic pulmonary fibrosis include a dry, non-productivecough on exertion; progressive exertional dyspnea; dry, inspiratorybibasilar crackles on auscultation; clubbing of the digits; and abnormalpulmonary function tests with evidence of restriction and impaired gasexchange.

The present invention further provides a method of treating orpreventing inflammation associated with post-surgical wound healing in apatient comprising administering to said patient a compound of theinvention.

It should be noted that compounds of the current invention may be usedto treat or prevent any disease which has an inflammatory component,such as those diseases cited above. Further, the inflammatory conditionscited above are meant to be exemplary rather than exhaustive.

Those skilled in the art would recognize that additional inflammatoryconditions (e.g., systemic or local immune imbalance or dysfunction dueto an injury, an insult, infection, inherited disorder, or anenvironmental intoxicant or perturbant to the subject's physiology) maybe treated or prevented by compounds of the current invention. Thus, themethods of the current invention may be used to treat or prevent anydisease which has an inflammatory component, including, but not limitedto, those diseases cited above.

In certain embodiments, the therapeutic compound disclosed hereindecreases one or more symptoms of inflammation by, e.g., at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% or at least 95%. In certain embodiments, the therapeuticcompound disclosed herein decreases one or more symptoms of inflammationby, e.g., about 5% to about 100%, about 10% to about 100%, about 20% toabout 100%, about 30% to about 100%, about 40% to about 100%, about 50%to about 100%, about 60% to about 100%, about 70% to about 100%, about80% to about 100%, about 10% to about 90%, about 20% to about 90%, about30% to about 90%, about 40% to about 90%, about 50% to about 90%, about60% to about 90%, about 70% to about 90%, about 10% to about 80%, about20% to about 80%, about 30% to about 80%, about 40% to about 80%, about50% to about 80%, or about 60% to about 80%, about 10% to about 70%,about 20% to about 70%, about 30% to about 70%, about 40% to about 70%,or about 50% to about 70%.

In certain embodiments, the therapeutic compound disclosed hereindecreases one or more symptoms of inflammation by e.g., at least atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% or at least 95% as compared to a patient notreceiving the same treatment. In certain embodiments, the therapeuticcompound disclosed herein decreases one or more symptoms of inflammationby, e.g., about 5% to about 100%, about 10% to about 100%, about 20% toabout 100%, about 30% to about 100%, about 40% to about 100%, about 50%to about 100%, about 60% to about 100%, about 70% to about 100%, about80% to about 100%, about 10% to about 90%, about 20% to about 90%, about30% to about 90%, about 40% to about 90%, about 50% to about 90%, about60% to about 90%, about 70% to about 90%, about 10% to about 80%, about20% to about 80%, about 30% to about 80%, about 40% to about 80%, about50% to about 80%, or about 60% to about 80%, about 10% to about 70%,about 20% to about 70%, about 30% to about 70%, about 40% to about 70%,or about 50% to about 70% as compared to a patient not receiving thesame treatment.

In certain embodiments, the therapeutic compound disclosed hereinreduces the duration of one or more symptoms of inflammation for atleast 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 2 years, 3 years, 5 years, 6 years, 7 years, 8 years, 9years, or 10 years as compared to a patient not receiving the sametreatment.

Fibrosis

The present compositions prevent the generation of fibrosis whilesimultaneously supporting wound healing. Recruitment of inflammatorycells and the subsequent laying down of extracellular matrix duringwound repair is a normal and healthy response to tissue damage as cellsin the vicinity of the wound become activated and migrate to fill thebreach. However, the general end point of repair is excessive and poorlyordered matrix deposition and fibrosis, which affects normal-tissuearchitecture and ultimately can disable proper functioning of tissues.This occurs on a macro scale as well as micro-scale. However, there isalso fibrosis that is independent of the inflammatory response, forexample, radiation induced fibrosis.

Wherever adult tissue is damaged, there is a massive influx ofleukocytes in order to prevent infection. However, along with theirinvolvement in innate immunity, leukocytes also release factors thatinfluence the behavior of other cells around them. It is known thatinflammatory cells secrete factors that stimulate fibroblast growth andmany studies have indicated that inflammation may be beneficial to therepair process. For example, an early study of the messenger RNAsexpressed by activated macrophages at a wound site indicatedtransforming growth factor (TGF), platelet-derived growth factor (PDGF),and TGF as growth factors that are delivered by recruited macrophages,and each one of these growth factors has been shown in some way or otherto be beneficial in wound healing. Many more such factors are releasedby one or more of the infiltrating leukocytic lineages and almost all ofthese factors will possibly have some positive effect on some aspect ofrepair, be it keratinocyte motility, fibroblast proliferation orcontraction, or the wound angiogenic response. However, leukocytes canalso be bad for repair and may actually promote fibrosis.

Every organ of the body can mount a repair response that generallyresults in a fibrotic lesion. Lung fibrosis as a result of chronicobstructive pulmonary disease and liver fibrosis because of hepatitisinfection are just two examples.

Thus, the present compositions may be administered to treat, ameliorate,delay the onset of, or decrease the extent of fibrosis in conditionssuch as pulmonary fibrosis, idiopathic pulmonary fibrosis, acuterespiratory distress syndrome, cystic fibrosis, non-cystic fibrosisbronchiectasis, cirrhosis, liver fibrosis (caused, for example bychronic viral hepatitis B or C), endomyocardial fibrosis, old myocardialinfarction, atrial fibrosis, mediastinal fibrosis (soft tissue of themediastinum), myelofibrosis (bone marrow), retroperitoneal fibrosis,progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn'sdisease, gastrointestinal fibrosis, keloid conditions,scleroderma/systemic sclerosis, arthofibrosis, peyronie's disease,dupuytren's contracture, oral submucous fibrosis, or adhesivecapsulitis. In a certain embodiment, the present compounds areadministered before, concurrent with, or after kidney dialysis, askidney dialysis is known to cause kidney fibrosis.

Symptoms of fibrosis include but are not limited to shortness of breath,a dry cough, a persistent cough with thick spit and mucous, wheezing,fatigue, unexplained weight loss, aching muscles and joints,breathlessness, repeated lung infections, inflamed nasal passages,greasy stinky stools, poor weight gain and growth, intestinal blockage,severe constipation, fibroids in the liver, inability to open mouth orlimited range, persistent diarrhea, rectal bleeding, urgent need to movebowels, abdominal cramp and pain, sensation of incomplete evacuation ofbowel, constipation, fever and fatigue, mouth sores, perineal disease,stiffness of joint, inability to straighten or flex joint, itchy skin,growing scar tissue on skin, lumpy or ridged scar tissue, hardening orskin or epithelial tissues, acid reflux, numbness, decreased or lack ofurine output, or hemorrhaging from death of intestinal tissue.

In certain embodiments, a therapeutic compound disclosed herein reducesone or more symptoms of fibrosis by, e.g., at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95%. In yet other aspects of this embodiment, a therapeuticcompound disclosed herein reduces the area affected by fibrosis by,e.g., about 5% to about 100%, about 10% to about 100%, about 20% toabout 100%, about 30% to about 100%, about 40% to about 100%, about 50%to about 100%, about 60% to about 100%, about 70% to about 100%, about80% to about 100%, about 10% to about 90%, about 20% to about 90%, about30% to about 90%, about 40% to about 90%, about 50% to about 90%, about60% to about 90%, about 70% to about 90%, about 10% to about 80%, about20% to about 80%, about 30% to about 80%, about 40% to about 80%, about50% to about 80%, or about 60% to about 80%, about 10% to about 70%,about 20% to about 70%, about 30% to about 70%, about 40% to about 70%,or about 50% to about 70%.

In certain embodiments, the therapeutic compound disclosed herein has adecreased area affected by fibrosis by e.g., at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95% as compared to a patient not receiving the same treatment. Incertain embodiments, the therapeutic compound disclosed herein has adecreased area affected by fibrosis by, e.g., about 5% to about 100%,about 10% to about 100%, about 20% to about 100%, about 30% to about100%, about 40% to about 100%, about 50% to about 100%, about 60% toabout 100%, about 70% to about 100%, about 80% to about 100%, about 10%to about 90%, about 20% to about 90%, about 30% to about 90%, about 40%to about 90%, about 50% to about 90%, about 60% to about 90%, about 70%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, or about60% to about 80%, about 10% to about 70%, about 20% to about 70%, about30% to about 70%, about 40% to about 70%, or about 50% to about 70% ascompared to a patient not receiving the same treatment.

In certain embodiments, the therapeutic compound disclosed herein delaysthe onset of symptoms of fibrosis by at least 1 week, 2 weeks, 3 weeks,4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years ascompared to a patient not receiving the same treatment.

In certain embodiments, a therapeutic compound disclosed herein reducesthe size of a wound by, e.g., at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90% or at least 95%. Inyet other aspects of this embodiment, a therapeutic compound disclosedherein reduces the size of a wound by, e.g., about 5% to about 100%,about 10% to about 100%, about 20% to about 100%, about 30% to about100%, about 40% to about 100%, about 50% to about 100%, about 60% toabout 100%, about 70% to about 100%, about 80% to about 100%, about 10%to about 90%, about 20% to about 90%, about 30% to about 90%, about 40%to about 90%, about 50% to about 90%, about 60% to about 90%, about 70%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, or about60% to about 80%, about 10% to about 70%, about 20% to about 70%, about30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

Wound Healing

Furthermore, in certain embodiments the therapeutic compound orcompositions disclosed herein are administered to treat, ameliorate,delay the onset of, or decrease the extent of an acute wound (such as aburn, sun exposure or radiation), e.g., dermal wounds and internalwounds, and chronic wounds such as a diabetic foot ulcer, venous legulcer, ulcerous tissue caused by repeated trauma to the body, orimpaired wound healing due to age. Thus, the compounds disclosed hereinmay treat a burn, a diabetic foot ulcer, a venous leg ulcer, ulceroustissue caused by repeated trauma to the body, or a chronic wound due toage.

In certain embodiments, the therapeutic compound disclosed herein has adecreased wound depth or area by e.g., at least at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90% or at least 95% as compared to a patient not receiving the sametreatment. In certain embodiments, the therapeutic compound disclosedherein has a decreased depth or area by, e.g., about 5% to about 100%,about 10% to about 100%, about 20% to about 100%, about 30% to about100%, about 40% to about 100%, about 50% to about 100%, about 60% toabout 100%, about 70% to about 100%, about 80% to about 100%, about 10%to about 90%, about 20% to about 90%, about 30% to about 90%, about 40%to about 90%, about 50% to about 90%, about 60% to about 90%, about 70%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, or about60% to about 80%, about 10% to about 70%, about 20% to about 70%, about30% to about 70%, about 40% to about 70%, or about 50% to about 70% ascompared to a patient not receiving the same treatment.

In certain embodiments, the therapeutic compound or compositionsdisclosed herein speeds the healing of the wound by at least 1 week, 2weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1year, 2 years, 3 years, 5 years, 6 years, 7 years, 8 years, 9 years, or10 years as compared to a patient not receiving the same treatment.

Cancer

The present methods may also be for the treatment of cancer by theinhibition of cancer cell growth, propagation, or metastases. The actualsymptoms associated with cancer are well known and can be determined bya person of ordinary skill in the art by taking into account one or morefactors, including, without limitation, the location of the cancer, thecause of the cancer, the severity of the cancer, and/or the tissue ororgan affected by the cancer. Those of skill in the art will know theappropriate symptoms or indicators associated with a specific type ofcancer and will know how to determine if an individual is a candidatefor treatment as disclosed herein.

Exemplary forms of cancer which may be treated by the subject methodsinclude, but are not limited to, leukemia, non-Hodgkin's lymphoma,prostate cancer, bladder cancer, lung cancer (including either smallcell or non-small cell cancer), colon cancer, kidney cancer, livercancer, breast cancer, cervical cancer, endometrial or other uterinecancer, ovarian cancer, skin cancer (e.g., melanoma), testicular cancer,cancer of the penis, cancer of the vagina, cancer of the urethra, gallbladder cancer, esophageal cancer, or pancreatic cancer. Additionalexemplary forms of cancer which may be treated by the subject methodsinclude, but are not limited to, cancer of skeletal or smooth muscle,stomach cancer, cancer of the small intestine, cancer of the salivarygland, anal cancer, rectal cancer, thyroid cancer, parathyroid cancer,pituitary cancer, and nasopharyngeal cancer. In one particularembodiment, the present methods include treatment of leukemias such asacute myeloid leukemia or acute lymphocytic leukemia. In anotherembodiment, the present methods include treatment of breast cancer, lungcarcinoma, prostate cancer, central nervous system cancer, melanoma,ovarian cancer, renal, and/or colon cancer. In one particular aspect,the present methods may treat the sequelae pediatric brain cancer, suchas the effects of whole-brain irradiation, and the prevention ofcognitive deficits due to brain tissue damage resulting from radiation.For instance, the present compositions may be administered prior to,during, or after brain irradiation to maintain cognitive function byprotecting normal brain tissue.

In certain embodiments, a therapeutic compound or compositions disclosedherein reduces the size of a tumor by, e.g., at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95%. In yet other aspects of this embodiment, a therapeuticcompound or compositions disclosed herein reduces the size of a tumorfrom, e.g., about 5% to about 100%, about 10% to about 100%, about 20%to about 100%, about 30% to about 100%, about 40% to about 100%, about50% to about 100%, about 60% to about 100%, about 70% to about 100%,about 80% to about 100%, about 10% to about 90%, about 20% to about 90%,about 30% to about 90%, about 40% to about 90%, about 50% to about 90%,about 60% to about 90%, about 70% to about 90%, about 10% to about 80%,about 20% to about 80%, about 30% to about 80%, about 40% to about 80%,about 50% to about 80%, or about 60% to about 80%, about 10% to about70%, about 20% to about 70%, about 30% to about 70%, about 40% to about70%, or about 50% to about 70%.

In certain embodiments, a cancer therapeutic disclosed herein is capableof reducing the number of cancer cells in an individual suffering from acancer by, e.g., at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% or at least 95% as compared to apatient not receiving the same treatment. In other aspects of thisembodiment, a cancer therapeutic is capable of reducing the number ofcancer cells in an individual suffering from a cancer by, e.g., about10% to about 100%, about 20% to about 100%, about 30% to about 100%,about 40% to about 100%, about 50% to about 100%, about 60% to about100%, about 70% to about 100%, about 80% to about 100%, about 10% toabout 90%, about 20% to about 90%, about 30% to about 90%, about 40% toabout 90%, about 50% to about 90%, about 60% to about 90%, about 70% toabout 90%, about 10% to about 80%, about 20% to about 80%, about 30% toabout 80%, about 40% to about 80%, about 50% to about 80%, or about 60%to about 80%, about 10% to about 70%, about 20% to about 70%, about 30%to about 70%, about 40% to about 70%, or about 50% to about 70% ascompared to a patient not receiving the same treatment.

In certain embodiments, a therapeutically effective amount of a cancertherapeutic disclosed herein reduces the cancer cell population and/ortumor cell size in an individual by, e.g., at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 100%. In other aspects of this embodiment, atherapeutically effective amount of a cancer therapeutic disclosedherein reduces or maintains a cancer cell population and/or tumor cellsize in an individual by, e.g., at most 10%, at most 15%, at most 20%,at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, at most 95% or at most 100%. In yetother aspects of this embodiment, a therapeutically effective amount ofa cancer therapeutic disclosed herein reduces or maintains a cancer cellpopulation and/or tumor cell size in an individual by, e.g., about 10%to about 100%, about 10% to about 90%, about 10% to about 80%, about 10%to about 70%, about 10% to about 60%, about 10% to about 50%, about 10%to about 40%, about 20% to about 100%, about 20% to about 90%, about 20%to about 80%, about 20% to about 20%, about 20% to about 60%, about 20%to about 50%, about 20% to about 40%, about 30% to about 100%, about 30%to about 90%, about 30% to about 80%, about 30% to about 70%, about 30%to about 60%, or about 30% to about 50%.

Dosage and Pharmaceutical Compositions

The present methods may prevent a disease or condition or one or moresymptoms of a disease or condition. As used herein, a therapeutic that“prevents” a disorder or condition refers to a compound that, in astatistical sample, reduces the occurrence of the disorder or conditionin the treated sample relative to an untreated control sample, or delaysthe onset or reduces the severity of one or more symptoms of thedisorder or condition relative to the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered or used as a pharmaceuticalcomposition comprising, for example, a compound of the invention and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil, or injectable organic esters. In a preferredembodiment, when such pharmaceutical compositions are for humanadministration, particularly for invasive routes of administration(i.e., routes, such as injection or implantation, that circumventtransport or diffusion through an epithelial barrier), the aqueoussolution is pyrogen-free, or substantially pyrogen-free. The excipientscan be chosen, for example, to effect delayed release of an agent or toselectively target one or more cells, tissues or organs. Thepharmaceutical composition can be in dosage unit form such as tablet,capsule (including sprinkle capsule and gelatin capsule), granule,lyophile for reconstitution, powder, solution, syrup, suppository,injection or the like. The composition can also be present in atransdermal delivery system, e.g., a skin patch.

A pharmaceutical composition disclosed herein may comprise a therapeuticcompound in an amount sufficient to allow customary administration to anindividual. In certain embodiments, a pharmaceutical compositiondisclosed herein may comprise, e.g., at least 5 mg, at least 10 mg, atleast 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, atleast 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mgof a therapeutic compound. In certain embodiments, a pharmaceuticalcomposition disclosed herein may comprise, e.g., at least 5 mg, at least10 mg, at least 20 mg, at least 25 mg, at least 50 mg, at least 75 mg,at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, atleast 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, atleast 900 mg, at least 1,000 mg, at least 1,100 mg, at least 1,200 mg,at least 1,300 mg, at least 1,400 mg, or at least 1,500 mg of atherapeutic compound. In yet other aspects of this embodiment, apharmaceutical composition disclosed herein may comprise in the rangeof, e.g., about 5 mg to about 100 mg, about 10 mg to about 100 mg, about50 mg to about 150 mg, about 100 mg to about 250 mg, about 150 mg toabout 350 mg, about 250 mg to about 500 mg, about 350 mg to about 600mg, about 500 mg to about 750 mg, about 600 mg to about 900 mg, about750 mg to about 1,000 mg, about 850 mg to about 1,200 mg, or about 1,000mg to about 1,500 mg. In still certain embodiments, a pharmaceuticalcomposition disclosed herein may comprise in the range of, e.g., about10 mg to about 250 mg, about 10 mg to about 500 mg, about 10 mg to about750 mg, about 10 mg to about 1,000 mg, about 10 mg to about 1,500 mg,about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 50 mg toabout 750 mg, about 50 mg to about 1,000 mg, about 50 mg to about 1,500mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about100 mg to about 750 mg, about 100 mg to about 1,000 mg, about 100 mg toabout 1,500 mg, about 200 mg to about 500 mg, about 200 mg to about 750mg, about 200 mg to about 1,000 mg, about 200 mg to about 1,500 mg,about 5 mg to about 1,500 mg, about 5 mg to about 1,000 mg, or about 5mg to about 250 mg.

A pharmaceutical composition disclosed herein may comprise a solvent,emulsion or other diluent in an amount sufficient to dissolve atherapeutic compound disclosed herein. In certain embodiments, apharmaceutical composition disclosed herein may comprise a solvent,emulsion or a diluent in an amount of, e.g., less than about 90% (v/v),less than about 80% (v/v), less than about 70% (v/v), less than about65% (v/v), less than about 60% (v/v), less than about 55% (v/v), lessthan about 50% (v/v), less than about 45% (v/v), less than about 40%(v/v), less than about 35% (v/v), less than about 30% (v/v), less thanabout 25% (v/v), less than about 20% (v/v), less than about 15% (v/v),less than about 10% (v/v), less than about 5% (v/v), or less than about1% (v/v). In certain embodiments, a pharmaceutical composition disclosedherein may comprise a solvent, emulsion or other diluent in an amount ina range of, e.g., about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70%(v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v),about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v)to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v),about 4% (v/v) to 40% (v/v), about 4% (v/v) to 30% (v/v), about 4% (v/v)to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to 50% (v/v),about 6% (v/v) to 40% (v/v), about 6% (v/v) to 30% (v/v), about 6% (v/v)to 20% (v/v), about 6% (v/v) to 10% (v/v), about 8% (v/v) to 50% (v/v),about 8% (v/v) to 40% (v/v), about 8% (v/v) to 30% (v/v), about 8% (v/v)to 20% (v/v), about 8% (v/v) to 15% (v/v), or about 8% (v/v) to 12%(v/v).

The final concentration of a therapeutic compound disclosed herein in apharmaceutical composition disclosed herein may be of any suitableconcentration. In certain embodiments, the final concentration of atherapeutic compound in a pharmaceutical composition may be atherapeutically effective amount. In certain embodiments, the finalconcentration of a therapeutic compound in a pharmaceutical compositionmay be, e.g., at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL,at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, atleast 1,000 mg/mL, or at least 1,200 mg/mL. In certain embodiments, theconcentration of a therapeutic compound disclosed herein in the solutionmay be, e.g., at most 1,000 mg/mL, at most 1,100 mg/mL, at most 1,200mg/mL, at most 1,300 mg/mL, at most 1,400 mg/mL, at most 1,500 mg/mL, atmost 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL. Incertain embodiments, the final concentration of a therapeutic compoundin a pharmaceutical composition may be in a range of, e.g., about0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL toabout 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mLto about 3,000 mg/mL, about 1,000 mg/mL to about 3,000 mg/mL, about 100mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about500 mg/mL to about 2,000 mg/mL, about 750 mg/mL to about 2,000 mg/mL,about 1,000 mg/mL to about 2,000 mg/mL, about 100 mg/mL to about 1,500mg/mL, about 250 mg/mL to about 1,500 mg/mL, about 500 mg/mL to about1,500 mg/mL, about 750 mg/mL to about 1,500 mg/mL, about 1,000 mg/mL toabout 1,500 mg/mL, about 100 mg/mL to about 1,200 mg/mL, about 250 mg/mLto about 1,200 mg/mL, about 500 mg/mL to about 1,200 mg/mL, about 750mg/mL to about 1,200 mg/mL, about 1,000 mg/mL to about 1,200 mg/mL,about 100 mg/mL to about 1,000 mg/mL, about 250 mg/mL to about 1,000mg/mL, about 500 mg/mL to about 1,000 mg/mL, about 750 mg/mL to about1,000 mg/mL, about 100 mg/mL to about 750 mg/mL, about 250 mg/mL toabout 750 mg/mL, about 500 mg/mL to about 750 mg/mL, about 100 mg/mL toabout 500 mg/mL, about 250 mg/mL to about 500 mg/mL, about 0.00001 mg/mLto about 0.0001 mg/mL, about 0.00001 mg/mL to about 0.001 mg/mL, about0.00001 mg/mL to about 0.01 mg/mL, about 0.00001 mg/mL to about 0.1mg/mL, about 0.00001 mg/mL to about 1 mg/mL, about 0.001 mg/mL to about0.01 mg/mL, about 0.001 mg/mL to about 0.1 mg/mL, about 0.001 mg/mL toabout 1 mg/mL, about 0.001 mg/mL to about 10 mg/mL, or about 0.001 mg/mLto about 100 mg/mL.

In certain embodiments, a therapeutically effective amount of atherapeutic compound disclosed herein generally is in the range of about0.001 mg/kg/day to about 100 mg/kg/day. In certain embodiments, aneffective amount of a therapeutic compound disclosed herein may be,e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1mg/kg/day, at least 1.0 mg/kg/day, at least 5.0 mg/kg/day, at least 10mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day. In certainembodiments, an effective amount of a therapeutic compound disclosedherein may be in the range of, e.g., about 0.001 mg/kg/day to about 10mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75mg/kg/day, about 0.001 mg/kg/day to about 100 mg/kg/day, about 0.001mg/kg/day to about 150 mg/kg/day, about 0.001 mg/kg/day to about 200mg/kg/day, about 0.001 mg/kg/day to about 250 mg/kg/day, about 0.001mg/kg/day to about 300 mg/kg/day, about 0.001 mg/kg/day to about 350mg/kg/day, about 0.001 mg/kg/day to about 400 mg/kg/day, about 0.001mg/kg/day to about 450 mg/kg/day, about 0.001 mg/kg/day to about 500mg/kg/day, about 0.001 mg/kg/day to about 550 mg/kg/day, about 0.001mg/kg/day to about 600 mg/kg/day, about 0.001 mg/kg/day to about 650mg/kg/day, about 0.001 mg/kg/day to about 700 mg/kg/day, about 0.001mg/kg/day to about 750 mg/kg/day, or about 0.001 mg/kg/day to about 800mg/kg/day. In yet other aspects of this embodiment, an effective amountof a therapeutic compound disclosed herein may be in the range of, e.g.,about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day toabout 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01mg/kg/day to about 75 mg/kg/day, about 0.01 mg/kg/day to about 100mg/kg/day, about 0.01 mg/kg/day to about 150 mg/kg/day, about 0.01mg/kg/day to about 200 mg/kg/day, about 0.01 mg/kg/day to about 250mg/kg/day, about 0.01 mg/kg/day to about 300 mg/kg/day, about 0.01mg/kg/day to about 350 mg/kg/day, about 0.01 mg/kg/day to about 400mg/kg/day, about 0.01 mg/kg/day to about 450 mg/kg/day, about 0.01mg/kg/day to about 500 mg/kg/day, about 0.01 mg/kg/day to about 550mg/kg/day, about 0.01 mg/kg/day to about 600 mg/kg/day, about 0.01mg/kg/day to about 650 mg/kg/day, about 0.01 mg/kg/day to about 700mg/kg/day, about 0.01 mg/kg/day to about 750 mg/kg/day, or about 0.01mg/kg/day to about 800 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/dayto about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1mg/kg/day to about 75 mg/kg/day, about 0.1 mg/kg/day to about 100mg/kg/day, about 0.1 mg/kg/day to about 150 mg/kg/day, about 0.1mg/kg/day to about 200 mg/kg/day, about 0.1 mg/kg/day to about 250mg/kg/day, about 0.1 mg/kg/day to about 300 mg/kg/day, about 0.1mg/kg/day to about 350 mg/kg/day, about 0.1 mg/kg/day to about 400mg/kg/day, about 0.1 mg/kg/day to about 450 mg/kg/day, about 0.1mg/kg/day to about 500 mg/kg/day, about 0.1 mg/kg/day to about 550mg/kg/day, about 0.1 mg/kg/day to about 600 mg/kg/day, about 0.1mg/kg/day to about 650 mg/kg/day, about 0.1 mg/kg/day to about 700mg/kg/day, about 0.1 mg/kg/day to about 750 mg/kg/day, or about 0.1mg/kg/day to about 800 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 10 mg/kg/day to about 15 mg/kg/day, about 10 mg/kg/dayto about 20 mg/kg/day, about 10 mg/kg/day to about 25 mg/kg/day, about10 mg/kg/day to about 30 mg/kg/day, about 10 mg/kg/day to about 35mg/kg/day, about 10 mg/kg/day to about 40 mg/kg/day, about 10 mg/kg/dayto about 45 mg/kg/day, about 10 mg/kg/day to about 50 mg/kg/day, about10 mg/kg/day to about 75 mg/kg/day, about 10 mg/kg/day to about 100mg/kg/day, about 10 mg/kg/day to about 150 mg/kg/day, about 10 mg/kg/dayto about 200 mg/kg/day, about 10 mg/kg/day to about 250 mg/kg/day, about10 mg/kg/day to about 300 mg/kg/day, about 10 mg/kg/day to about 350mg/kg/day, about 10 mg/kg/day to about 400 mg/kg/day, about 10 mg/kg/dayto about 450 mg/kg/day, about 10 mg/kg/day to about 500 mg/kg/day, about10 mg/kg/day to about 550 mg/kg/day, about 10 mg/kg/day to about 600mg/kg/day, about 10 mg/kg/day to about 650 mg/kg/day, about 10 mg/kg/dayto about 700 mg/kg/day, about 10 mg/kg/day to about 750 mg/kg/day, orabout 10 mg/kg/day to about 800 mg/kg/day.

In other aspects of this embodiment, an effective amount of atherapeutic compound disclosed herein may be in the range of, e.g.,about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 15mg/kg/day, about 1 mg/kg/day to about 20 mg/kg/day, about 1 mg/kg/day toabout 25 mg/kg/day, about 1 mg/kg/day to about 30 mg/kg/day, about 1mg/kg/day to about 35 mg/kg/day, about 1 mg/kg/day to about 40mg/kg/day, about 1 mg/kg/day to about 45 mg/kg/day, about 1 mg/kg/day toabout 50 mg/kg/day, about 1 mg/kg/day to about 75 mg/kg/day, or about 1mg/kg/day to about 100 mg/kg/day. In certain embodiments, an effectiveamount of a therapeutic compound disclosed herein may be in the rangeof, e.g., about 5 mg/kg/day to about 10 mg/kg/day, about 5 mg/kg/day toabout 15 mg/kg/day, about 5 mg/kg/day to about 20 mg/kg/day, about 5mg/kg/day to about 25 mg/kg/day, about 5 mg/kg/day to about 30mg/kg/day, about 5 mg/kg/day to about 35 mg/kg/day, about 5 mg/kg/day toabout 40 mg/kg/day, about 5 mg/kg/day to about 45 mg/kg/day, about 5mg/kg/day to about 50 mg/kg/day, about 5 mg/kg/day to about 75mg/kg/day, or about 5 mg/kg/day to about 100 mg/kg/day.

In liquid and semi-solid formulations, a concentration of a therapeuticcompound disclosed herein typically may be between about 50 mg/mL toabout 1,000 mg/mL. In certain embodiments, a therapeutically effectiveamount of a therapeutic disclosed herein may be from, e.g., about 50mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50mg/mL to about 900 mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100mg/mL to about 400 mg/mL, about 100 mg/mL to about 500 mg/mL, about 100mg/mL to about 600 mg/mL, about 100 mg/mL to about 700 mg/mL, about 100mg/mL to about 800 mg/mL, about 100 mg/mL to about 900 mg/mL, about 100mg/mL to about 1,000 mg/mL, about 200 mg/mL to about 300 mg/mL, about200 mg/mL to about 400 mg/mL, about 200 mg/mL to about 500 mg/mL, about200 mg/mL to about 600 mg/mL, about 200 mg/mL to about 700 mg/mL, about200 mg/mL to about 800 mg/mL, about 200 mg/mL to about 900 mg/mL, about200 mg/mL to about 1,000 mg/mL, about 300 mg/mL to about 400 mg/mL,about 300 mg/mL to about 500 mg/mL, about 300 mg/mL to about 600 mg/mL,about 300 mg/mL to about 700 mg/mL, about 300 mg/mL to about 800 mg/mL,about 300 mg/mL to about 900 mg/mL, about 300 mg/mL to about 1,000mg/mL, about 400 mg/mL to about 500 mg/mL, about 400 mg/mL to about 600mg/mL, about 400 mg/mL to about 700 mg/mL, about 400 mg/mL to about 800mg/mL, about 400 mg/mL to about 900 mg/mL, about 400 mg/mL to about1,000 mg/mL, about 500 mg/mL to about 600 mg/mL, about 500 mg/mL toabout 700 mg/mL, about 500 mg/mL to about 800 mg/mL, about 500 mg/mL toabout 900 mg/mL, about 500 mg/mL to about 1,000 mg/mL, about 600 mg/mLto about 700 mg/mL, about 600 mg/mL to about 800 mg/mL, about 600 mg/mLto about 900 mg/mL, or about 600 mg/mL to about 1,000 mg/mL.

As used herein, “mitigating” means reducing one or more negativesymptoms of a condition, relative to a cell, organ, tissue, or organismdisplaying the symptom or condition for the same amount of time, butuntreated.

In some embodiments, contacting the cell, organ, tissue, or organism thepresent compounds may comprise administering a therapeutically effectiveamount of the compound to a subject. As used herein, a “therapeuticallyeffective amount” is an amount sufficient to mitigate the negativesymptom or condition.

The subject may be a human, rat, mouse, cat, dog, horse, sheep, cow,monkey, avian, or amphibian. In another embodiment, the cell is in vivoor in vitro. Typical subjects to which compounds of the invention may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e. g. livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use such as mammalian, particularlyprimate such as human, blood, urine or tissue samples, or blood urine ortissue samples of the animals mentioned for veterinary applications.

The cell, organ, tissue, or organism may be contacted with a compounddescribed herein before, during, or after evidencing symptoms of thecondition or disease, or before the predicate event leading to anexpected condition or disease. In some embodiments, the compound may beadministered prophylactically, e.g., where radiation-inducedthrombocytopenia is expected, before the predicate event of exposure toionizing radiation, for example, prior to cancer radiation therapy orX-ray, or prior to development of fibrosis in advanced HIV. In someembodiments, the compound may be administered during the predicateevent, or upon repeated exposure to the predicate event. In someembodiments, the compound may be administered after the predicate event,such as after exposure to ionizing radiation, or after the initiation ofexposure to radiation.

When administering to an organism, the compound may be administered byany suitable means. In some embodiments, the compounds or formulationsare administered orally. In some embodiments, the compounds orformulations are administered by injection, e.g. subcutaneous,parenteral, or intravenous, injections.

In some embodiments, the compound may be administered in combinationwith other potential mitigators or with other toxic agents such as thechemotherapeutic drugs discussed above. In a particular embodiment, thecomposition may be administered with growth factors, NSAIDs,chemotherapeutics, anti-inflammatories, antibiotics, Metformin(Glucophage, Glumetza, others), Sulfonylureas, Meglitinides,Thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2inhibitors, and/or Insulin therapy, for the treatment of the aboveconditions. In one aspect, the growth factor can be G-CSF (akafilgrastim, NEUPOGEN®) or erythropoietin (aka EPOGEN®).

In other embodiments, the compositions may comprise an effective amountof a modulator and/or other pharmaceutically active agent in aphysiologically-acceptable carrier. The carrier may take a wide varietyof forms depending on the form of preparation desired for a particularroute of administration. Suitable carriers and their formulation aredescribed, for example, in Remington's Pharmaceutical Sciences by E. W.Martin.

In some embodiments, the compound may be contained in any appropriateamount in any suitable carrier substance, and is generally present in anamount of 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable forparenteral (e.g., subcutaneously, intravenously, intramuscularly, orintraperitoneally) or oral administration route. The pharmaceuticalcompositions may be formulated according to conventional pharmaceuticalpractice (see, e.g., Remington: The Science and Practice of Pharmacy(20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 andEncyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C.Boylan, 1988-1999, Marcel Dekker, New York).

In some embodiments, the compositions may be in a form suitable foradministration by sterile injection. In one example, to prepare such acomposition, the compositions(s) are dissolved or suspended in aparenterally acceptable liquid vehicle. Among acceptable vehicles andsolvents that may be employed are water, water adjusted to a suitable pHby addition of an appropriate amount of hydrochloric acid, sodiumhydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, andisotonic sodium chloride solution and dextrose solution. The aqueousformulation may also contain one or more preservatives (e.g., methyl,ethyl or n-propyl p-hydroxybenzoate). For parenteral formulations, thecarrier will usually comprise sterile water, though other ingredients,for example, ingredients that aid solubility or for preservation, may beincluded. Injectable solutions may also be prepared in which caseappropriate stabilizing agents may be employed. In one embodiment, theformulation includes at least one or more of methanesulfonic acid,povidone, benzyl alcohol, n-Methyl pyrrolidone, ethaonol, Poloxamer 188,lactic acid, Captisol (SBE-beta-CD), or Vitamin E, such as TPGS (d-alphatocopheryl polyethylene glycol 1000 succinate).

Formulations suitable for parenteral administration usually comprise asterile aqueous preparation of the compound, which may be isotonic withthe blood of the recipient (e.g., physiological saline solution). Suchformulations may include suspending agents and thickening agents andliposomes or other microparticulate systems which are designed to targetthe compound to blood components or one or more organs. The formulationsmay be presented in unit-dose or multi-dose form.

Parenteral administration may comprise any suitable form of systemicdelivery or localized delivery. Administration may for example beintravenous, intra-arterial, intrathecal, intramuscular, subcutaneous,intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may beeffected by infusion pumps (external or implantable) or any othersuitable means appropriate to the desired administration modality.

In some embodiments, the compositions may be in a form suitable for oraladministration. In compositions in oral dosage form, any of the usualpharmaceutical media may be employed. Thus, for liquid oralpreparations, such as, for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like. For solidoral preparations such as, for example, powders, capsules and tablets,suitable carriers and additives include starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike. If desired, tablets may be sugar coated or enteric coated bystandard techniques.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the active ingredient as a powderor granules. Optionally, a suspension in an aqueous liquor or anon-aqueous liquid may be employed, such as a syrup, an elixir, anemulsion, or a draught. Formulations for oral use include tabletscontaining active ingredient(s) in a mixture with pharmaceuticallyacceptable excipients. Such formulations are known to the skilledartisan. Excipients may be, for example, inert diluents or fillers(e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose,starches including potato starch, calcium carbonate, sodium chloride,lactose, calcium phosphate, calcium sulfate, or sodium phosphate);granulating and disintegrating agents (e.g., cellulose derivativesincluding microcrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

A syrup may be made by adding the compound to a concentrated aqueoussolution of a sugar, for example sucrose, to which may also be added anyaccessory ingredient(s). Such accessory ingredient(s) may includeflavorings, suitable preservative, agents to retard crystallization ofthe sugar, and agents to increase the solubility of any otheringredient, such as a polyhydroxy alcohol, for example glycerol orsorbitol.

In some embodiments, the composition may be in a form of nasal or othermucosal spray formulations (e.g. inhalable forms). These formulationscan include purified aqueous solutions of the active compounds withpreservative agents and isotonic agents. Such formulations can beadjusted to a pH and isotonic state compatible with the nasal or othermucous membranes. Alternatively, they can be in the form of finelydivided solid powders suspended in a gas carrier. Such formulations maybe delivered by any suitable means or method, e.g., by nebulizer,atomizer, metered dose inhaler, or the like.

In some embodiments, the composition may be in a form suitable forrectal administration. These formulations may be presented as asuppository with a suitable carrier such as cocoa butter, hydrogenatedfats, or hydrogenated fatty carboxylic acids.

In some embodiments, the composition may be in a form suitable fortransdermal administration. These formulations may be prepared, forexample, by incorporating the active compound in a thixotropic orgelatinous carrier such as a cellulosic medium, e.g., methyl celluloseor hydroxyethyl cellulose, with the resulting formulation then beingpacked in a transdermal device adapted to be secured in dermal contactwith the skin of a wearer.

In addition to the aforementioned ingredients, compositions of theinvention may further include one or more accessory ingredient(s)selected from encapsulants, diluents, buffers, flavoring agents,binders, disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like.

In some embodiments, compositions may be formulated for immediaterelease, sustained release, delayed-onset release or any other releaseprofile known to one skilled in the art.

In some embodiments, the pharmaceutical composition may be formulated torelease the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or inthe central nervous system or cerebrospinal fluid; (v) formulations thatallow for convenient dosing, such that doses are administered, forexample, once every one or two weeks; and (vi) formulations that targetthe site of a pathology. For some applications, controlled releaseformulations obviate the need for frequent dosing to sustain activity ata medically advantageous level.

In a certain embodiment or a particular formulation BCN057 wassolubalized in aqueous solution at physiologically compatible pHs using100 mM methanesulfonic acid (MSA)/10% povidone (PVP); 100 mM MSA/2%benzyl alcohol/2% N-methylpyrrolidone (NMP); and, 100 mM MSA/10%ethanol/1% Poloxamer 188. In a further aspect 100 mM lactic acid wasadded and also improved solubility for these mixtures. In yet anotherembodiment, a formulation comprising BCN057 and 30 wt % Captisol(SBE-beta-CD) and 100 mM MSA yielded excellent solubility at up to pH4.1 or higher.

In another embodiment formulation for intravenous, subcutaneous and oraldelivery of therapeutic levels of BCN057 were developed comprising 30 wt% Captisol (SBE-beta-CD) and 100 mM MSA at pH 4.1 or higher (adjustedwith 1.0 N NaOH).

In one embodiment for 512, formulations containing DMA(dimethylacetimide) or DMSO or Polyvinylepyrolidone are used.Suspensions (micron or nano diameter particle sizes) are useful for thedrug since it wants to self-associate and crash out otherwise.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the compound is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the compound in a controlled manner. Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

In some embodiments, the composition may comprise a “vectorized” form,such as by encapsulation of the compound in a liposome or otherencapsulate medium, or by fixation of the compound, e.g., by covalentbonding, chelation, or associative coordination, on a suitablebiomolecule, such as those selected from proteins, lipoproteins,glycoproteins, and polysaccharides.

In some embodiments, the composition can be incorporated intomicrospheres, microcapsules, nanoparticles, liposomes, or the like forcontrolled release. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents. Alternatively, the compound may beincorporated in biocompatible carriers, implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

In all embodiments, the compound or other active compounds may bepresent as pharmaceutically acceptable salts or other derivatives, suchas ether derivatives, ester derivatives, acid derivatives, and aqueoussolubility altering derivatives of the active compound. Derivativesinclude all individual enantiomers, diastereomers, racemates, and otherisomers of the compounds. Derivatives also include all polymorphs andsolvates, such as hydrates and those formed with organic solvents, ofthe compounds. Such isomers, polymorphs, and solvates may be prepared bymethods known in the art, such as by regiospecific and/orenantioselective synthesis and resolution.

The ability to prepare salts depends on the acidity or basicity of thecompounds. Suitable salts of the compounds include, but are not limitedto, acid addition salts, such as those made with hydrochloric,hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic, cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as sodium and potassiumsalts; alkaline earth metal salts, such as calcium and magnesium salts;and salts formed with organic or inorganic ligands, such as quaternaryammonium salts.

Additional suitable salts include, but are not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammoniumsalt, oleate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide and valerate salts of the compounds.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Unless the context clearly indicates otherwise, compositions of allembodiments can comprise various pharmaceutically acceptable salts, orother derivatives described above.

The formulation and preparation of such compositions are well known tothose skilled in the art of pharmaceutical formulation. Formulations canbe found in Remington: The Science and Practice of Pharmacy.

The amount of the compound employed in the present invention to be usedvaries according to the condition, the patient/subject, and the extentof the condition.

The contents of all cited references (including literature references,issued patents, published patent applications) as cited throughout thisapplication are hereby expressly incorporated by reference. Theinvention and the manner and process of making and using it, aredescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame.

The term “unit dosage form” or “unit” as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of thecompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable, diluent,carrier or vehicle. The specifications for the novel unit dosage formsof the present invention depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the subject.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent. Methods todetermine efficacy and dosage are known to those skilled in the art(Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

Dosing can be single dosage or cumulative (serial dosing), and can bereadily determined by one skilled in the art. For instance, treatmentmay comprise a one-time administration of an effective dose of apharmaceutical composition disclosed herein. Alternatively, treatmentmay comprise multiple administrations of an effective dose of apharmaceutical composition carried out over a range of time periods,such as, e.g., once daily, twice daily, thrice daily, once every fewdays, or once weekly. The timing of administration can vary fromindividual to individual, depending upon such factors as the severity ofan individual's symptoms. For example, an effective dose of apharmaceutical composition disclosed herein can be administered to anindividual once daily for an indefinite period of time, or until theindividual no longer requires therapy. A person of ordinary skill in theart will recognize that the condition of the individual can be monitoredthroughout the course of treatment and that the effective amount of apharmaceutical composition disclosed herein that is administered can beadjusted accordingly.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

In certain embodiments, the period of administration of a therapeuticcompound is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, or more. In certain embodiments, atreatment regimen may comprise a period during which administration isstopped for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, or more.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In other embodiments, the compounds described herein may be providedwith the one or more additional therapeutic agents in a kit, e.g., asseparate pharmaceutical formulations capable of being used together in aconjoint therapy as discussed herein, either together in a singlecontainer or in separate containers. In certain such embodiments, thekit may further include instructions for the conjoint administration ofthe pharmaceutical formulations, e.g., for treating or preventing any ofthe conditions discussed above.

Such combination products may employ compounds of this invention, orpharmaceutically acceptable salts thereof, within the dosage rangedescribed hereinbefore and the other pharmaceutically-active agentwithin its approved dosage range.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Example 1: Cancer Cell Growth Inhibition

A cancer cell survival profile on multiple leukemia cell lines wasconducted on BCN057 as described in Monks, A.; Scudiero, D. A.; Skehan,P.; Shoemaker, R. H.; Paull, K. D.; Vistica, D. T.; Hose, C.; Langley,J.; Cronice, P.; Vaigro-Wolf, M.; Gray-Goodrich, M.; Campbell, H.; Mayo,M. R. JNCI, J. Natl. Cancer Inst. 1991, 83, 757-766. The assay showspercent growth over 48 h at 10 uM BCN057 vs control (no drug). The drugwas inhibitory towards Leukemia and also towards prostate and kidneycancer (data not shown). Examples of cell lines include: SR; large cellimmunoblastic lymphoma, RPMI-8226; plasmacytoma and myeloma, MOLT-4;acute T lymphoblastic leukemia, K562; erythromyeloblastoid leukemia orchronic myeloid leukemia cell line, HL-60 (TB); acute myeloid leukemia,CCRF-CEM; T cell lymphoblast-like cell line. Results are shown in FIG. 1for RPMI-8226, K-562, and CCRF-CEM

In addition to leukemia and lymphomas, other tumor cell lines weresusceptible to the BCN057 such as breast cancer, lung carcinoma,prostate, central nervous system (CNS), melanoma, ovarian, prostate, andrenal and colon cancer (FIG. 2).

The experiment was further conducted on the breast cancer cell lines:T-470, MDA-MB-231/ATCC, H5578T, BT-549, and MDA-MB-468; the CNS cancerlines: SNB-19, SNB-75, SF-539; Colon cancer cell line HCT-116; Melanomacell lines: UACC-62 SK-MEL-5; non-small cell lung cancer cell lines:HOP-92, EKVX, NCI-H23; ovarian cancer cell lines IGROV1, NCI/ADR-RES,OVCAR-4; prostate cancer cell line PC-3; and renal cancer cell lines:A498, ACHN, UO-31, CAKI-1, and 786Q. Results are shown in FIG. 2, wherethe Y-axis represents percent growth relative to a control of the samecell type untreated (i.e., 100% in each case)

It is demonstrated that BCN057 causes significant inhibition of thecancer cell growth, showing a direct effect on the cancer itself, andnot only the conditions caused by treatments for cancer. This effect isunexpected for this compound.

In vitro studies have shown the drugs can inhibit cancer cellproliferation. The cancers affected include renal cancer, prostatecancer, non-small cell lung cancer, breast cancer, colon cancer,ovarian, leukemia, skin cancer such as melanoma, central nervous systemcancers including pediatric brain cancers and adult brain cancers.Within this class of cancers, in particular the drugs show importantinhibition of epithelial cancers such as colon, breast and oral cancerswhile protecting normal tissue such as in oral mucositis, proctitis andmucositis of the intestine.

Example 2: Effect on Platelet Count

Normal mice were treated with BCN057 for eight days by oraladministration or subcutaneous injection. Dose groups included 75mg/kg/day, 200 mg/kg/day, 400 mg/kg/day, 500 mg/kg/day, and 800mg/kg/day

BCN057 induces platelet production in a dose dependent fashion. FIG. 3is a graph of the amount of blood platelets from plasma (mouse)demonstrating through multiple doses and multiple routes of entry thatthe drug stimulates platelet production rapidly.

Example 3: Restoration of Cytokines after Irradiation

Mice were irradiated on Day 0. Animals were dosed with drug for 7consecutive days (Day 1 to 7) at 200 mg/kg SC, with terminal bloodcollection on Day 8. Plasma from 3 mice was pooled for each condition Yaxis represents relative absorbance units.

The plasma was tested with the Mouse Cytokine panel ELISA Array panel—bySIGNOSIS™

Results are shown in FIGS. 4 and 5. There is a general trend ofrestoring cytokines to levels similar to control with exceptions. PDGFis important for restoration of mesenchyme and endothelial cells alongwith FGF. IL-6 and IL-10 are anti-inflammatory cytokines while GCSF andGMCSF affect macrophage infiltration and activation. FIG. 5 shows thecytokine analysis of plasma from mice treated with nothing (marked as 0Gabove), 7 Grey radiation (7G) and 7G+BCN057). A restorative phenotype isobserved in the presence of the drug similar to that of the controlgroup receiving no radiation vs the group receiving radiation alone.This is important for inflammatory disorders as well as fibroticdisorders

BCN057 alters key cytokines in blood to prevent an inflammatorycondition. Analysis of the cytokines in animals treated with the drugafter injury by radiation for example; there is a general theme ofrestoring cytokines to levels similar to control with some exceptions.IGF: known implications in gastrointestinal inflammatory diseases PDGFis associated with wound healing and tissue repair. Known to restoreenterocytes and intestinal cell replacement. PDGF is also known torestore mesenchyme and endothelial cells as well. PDGF levels being highin the presence of the drug may also explain platelet production. FGF:restoration of mesenchyme and endothelial cells. IL-6 and IL-10 areanti-inflammatory cytokines involved in amelioration of sepsis followingGI radiation injury or GI inflammatory disorders. GCSF and GMCSF promoteor induce macrophage infiltration and activation in intestine. EGF is anintestinal epithelial growth factor. In most cases, BCN057 restores thecytokine profile to similar to that of the control on day 1.

Example 4: Hematopoietic Recovery Following Irradiation

Recovery of the hematopoietic system following 6 Gy irradiation of C3Hmice (n=4) with BCN057 treatment (s.c.) at 24, 48, 72, 96, and 120 hrs.RBC—red blood cells (M/uL), HB-hemoglobin (g/dL), and HCT-hematocrit(%). B: recovery of the hematopoietic system following 6 Gy irradiationof C3H mice (n=4) with BCN057 treatment (s.c.) at 24, 48, 72, 96, and120 hrs. WBC-white blood cells, NE-neutrophils, LY-lymphocytes. C:platelet (PLT) recovery following 6 Gy irradiation of C3H mice (n=4)with BCN057 treatment (s.c.) at 24,48,72,96, and 120 hrs; p<0.05 with a1-tail, Student t-test. The drug shows evidence of improvinghematopoietic recovery. See FIGS. 8 to 10.

Example 5: Formulations

Several formulations were developed to solubilize BCN057 in aqueoussolution at physiologically compatible pHs. These include 100 mMmethanesulfonic acid/10% povidone (PVP); 100 mM MSA/2% benzyl alcohol/2%N-methylpyrrolidone (NMP); and, 100 mM MSA/10% ethanol/1% Poloxamer 188.The addition of 100 mM lactic acid also improved solubility for thesemixtures. Also, solutions containing 30 wt % Captisol (SBE-beta-CD) and100 mM MSA yielded excellent solubility at up to pH 4.1.

Several suitable formulations were developed for intravenous,subcutaneous and oral delivery of therapeutic levels of BCN057. Theseinclude 30 wt % Captisol (SBE-beta-CD) and 100 mM MSA at pH 4.1(adjusted with 1.0 N NaOH).

Furthermore, analytical methods to determine purity and quantity in drugproduct were created which included HPLC Assay and Impurities using areverse-phase gradient method with C18 column and UV detection at 210nm; osmolality and pH using standard techniques.

Example 6: BCN512 and Lung Fibrosis

Our goal is to develop a drug to protect lung from ionizing radiationthat is administered subcutaneously and has a favorable risk/benefitprofile when administered 24 hours or later after radiation exposure.The drug, BCN 512, originally discovered in the UCLA CMCR, is a noveldrug that emerged from high-throughput screening of small moleculelibraries, and is now under investigation as a radiation injurymitigator. In our efforts to develop the drug as a lung MCM to treat theDelayed Effects of Acute Radiation Exposure (DEARE), we use the C57BL/6and C3H mouse models. These models appear to recapitulate human lungfibrosis and pneumonitis, respectively, based upon the distinctsensitivity of the two strains (1, 2). Survival and histology datasuggest that in these rodent models, BCN512 is effective in amelioratinglung fibrosis and pneumonitis.

Data: Mice (8 per group) exposed to single whole thoracic lungirradiation (18 Gy at 0.6 Gy/min using an AEC Gammacell® 40 Cs-137source) subcutaneous administrations of 512 in Cremophor on days 1-5post-irradiation at 5 mg/Kg improved overall survival from pneumonitisand lung fibrosis at 90 days and 160 respectively. Radiation fibrosis isa progressive, dose-related, late complication of radiation exposure,with animals and humans surviving for some time with non-lethal damage.FIG. 18 shows that after 14 Gy, which is not lethal for most mice, theacute delivery of BCN512 mitigates against the development of this latedisease. 512 works by altering the inflammatory infiltrate into thelung. By day 160 after LTI the mature macrophage cell content is greatlydecreased as a result of acute drug treatment.

Because the side effects of Cremophor are undesirable for an MCMcandidate, alternative formulations are being tested for furtherdevelopment of 512 for the lung DEARE indication. Once such formulationusing Deoxycholate shows plasma exposure (FIG. 6A) when givensubcutaneously (SC) and also shows good efficacy in total bodyirradiation experiments indicating that the drug has retained itsmitigation properties.

In addition, other important prototype formulations are being developedin partnership with Particle Sciences, Bethlehem, Pa., to createnanoparticle formulations of the drug substance. Furthermore,nanoparticle drug substances are known to more preferentially distributeto sites of inflammation (3) which may be favorable in this case

512 is a lead candidate for the development of a drug for delayedeffects of radiation exposure in lung. Further work will includeextensive testing in lung models along with characterization of thetoxicology, pharmacology and metabolism of the drug product beforepivotal non GLP animal studies.

Example 7: Radio-Mitigation of Normal but not Tumor Tissue

One of the major concerns of the Food and Drug Administration for theapplication of radiation mitigators in RT is that if an agent canprotect normal tissue from radiation damage, it may also protect tumortissues. In our phase I proposal, we used syngeneic, allogeneic, andxenograft lung tumor models to show that the drug did not accelerate thegrowth of tumor lung colonies in vivo with or without LTI (FIG. 3A-B).The syngeneic model of artificial metastasis used Lewis lung (LLC)tumors, and since LLC also grow in C3H mice, this could be employed asan allogeneic model. C57Bl/6 and C3H mice were injected i.v. with 5×104tumor cells. Subcutaneous drug injections were started on day +3 whenthe tumors were established in the lung in order to bias the experimentin favor of tumor growth promotion. The dose regimen was arbitrarilyassigned to a dose of 20 mg/kg for 5 days. LTI was started on day 4,with 4 Gy doses administered daily for 3 days. This is higher thanconventional 2 Gy to compensate for the more rapid growth of murinetumors, but is still well within the range used clinically inhypofractionated therapy.

Treatment with BCN512 significantly (P<0.05) decreased the number oflung tumor colonies on day 14 by 20% in both C3H and C57 mouse strains,and the colonies in both strains were smaller in size than control. LTIalone decreased the number of co DNA repair lonies by 40%. Extensiveanalysis of the drug doses that would be optimal for exerting effects ontumors in the radiation setting are still needed, but there is noevidence for enhanced tumor growth as a result of radiation treatmentwith drug, and in fact exactly the opposite. We are therefore confidentthat BCN512 shows anti-tumor activity in vivo. FIG. 3C shows additionaldata from the A549 human NSCLC cell line. Thirty-two nude mice wereinjected intravenously with 5×10⁴ human A549 adenocarcinoma cells onday 1. The drug was administered starting at day 3; 20 mg/kg BCN512 wasinjected subcutaneously once daily for 5 days. Fractionated radiationwas started on day 4, with 4 Gy LTI administered daily for 3 days.Tumors developed very slowly, so the mice were sacrificed on day 72 andthe number of nodules in the lungs were counted. There was considerablevariation in the irradiated group, with a tendency for lung irradiationto increase the number of tumor colonies. This is not a uniqueobservation that may be ascribed to radiation-induced myeloid cellmobilization. In any event, BCN512 did not increase the number of tumorcolonies, and if anything decreased the count, especially in theirradiated group.

See FIG. 6B. The NCI screening procedures were as described (1) as werethe origins and processing of the cell lines (1, 2, 3, 4). Briefly, cellsuspensions that were diluted according to the particular cell type andthe expected target cell density (5000-40,000 cells per well based oncell growth characteristics) were added by pipet (100 μL) into 96-wellmicrotiter plates. Inoculates were allowed a preincubation period of 24h at 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100-μ L aliquots to themicrotiter plate wells. Usually, test compounds were evaluated at five10-fold dilutions. In routine testing, the highest well concentration is1×10−4 M, but for the standard agents the highest well concentrationused depended on the agent. Incubations lasted for 48 h in 5% CO2atmosphere and 100% humidity. The cells were assayed by using thesulforhodamine B assay (5, 6). A plate reader was used to read theoptical densities, and a microcomputer processed the optical densitiesinto the special concentration parameters defined later. ScreeningProcedures Leukemia; CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR.Non-Small Cell Lung Cancer; 549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226,NCI-H23, NCI-H322M, NCI-H460, NCI-H522. Colon Cancer; COLO 205,HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620. CNS Cancer; SF-268,SF-295, SF-539, SNB-19, SNB-75, U251. Melanoma; LOX IMVI, MALME-3M, M14,MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62. OvarianCancer; IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES,SK-OV-3. Renal Cancer; 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10,UO-31. Prostate Cancer; PC-3, DU-145. Breast Cancer; MCF7,MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468.

Example 8: Hematopoiesis

Both BCN057 and BCN512 activate Wnt signaling in stem cells promotingself-renewal and proliferation. Hematopoietic stem cells are also drivenby wnt signaling and are thus stimulated by the drug.

FIGS. 8 A-B show that BCN057 helps prevent hematopoietic suppressionfrom total body irradiation in male and female mice. 7.75 Gy radiationis used to ablate bone marrow hematopoietic stem cells (HSCs) in thesestrains of mice. BCN057 is sufficient to prevent lethality from bonemarrow suppression. Lethality from bone marrow suppression occurs in the14-25 day region due to the life cycle of approximately 2 weeks for RBCand inability to replace them without HSCs

FIG. 8 C shows that bone marrow-derived stem cells express hematopoieticcytokines IL3, 116, IL11, GCSF, GMCSF, LIF, MCSF, SCF, which areimportant for supporting long-term hematopoiesis. Plasma levels of, 116,GCSF, GMCSF, SCF are elevated after drug treatment and in conjunctionwith the radiation treatment indicating hematopoietic stem cells areeffected

FIG. 9 shows that BCN 512 prevents hematopoietic suppression from totalbody irradiation in mice. 7.73 Gy radiation is used to ablate bonemarrow HSCs in the C3H strain of mice. BCN512 is sufficient to preventlethality from bone marrow suppression from total body irradiation.

FIG. 10 shows that radiation ablates the bone marrow which exhibits aspale bone marrow devoid of RBC's (LTI). Finally, radiation (LTI) andBCN512 and LTI (last to right) show similar red color (hemoglobin) tocontrol.

In summary, whole body irradiation studies are used to ablate thehematopoietic system (red blood cells, white blood cells and theirprogenitors along with stem cells from which they are all derived) inboth humans and mammalian bone marrow. The bone marrow will become whitedue to a-cellularity and become unable to produce adequate red and whitecells with a resulting death at a radiation dose that is specific forthe hematopoietic system (other organs are not appreciably affected).The survival associated with these drugs without blood replacement alongwith the presence in plasma of bone marrow derived cytokines and/orplatelet increase associated with the presence of the cells in thetreated group vs the untreated group show the drugs promotehematopoiesis and prevent neutropenia in the case of insult to bonemarrow by toxic agents.

Example 9: Fibrosis and Wound Healing

512 activates canonical Wnt-β catenin signaling: To determine thecanonical Wnt activity induced by BCN-512 (28 μM), HEK293 cellspossessing a TCF/LEF luciferase reporter construct were treated withBCN-512 or vehicle control. LiCl (10 mM) treatment was used as positivecontrol for luciferase activity. Luciferase activity was determinedafter 24 h using a Dual-Luciferase® Reporter Assay System (Promega) asper manufacturer's protocol. HEK293 cells containing a FOPFlashconstruct were used as a negative control. BCN-512 treatmentsignificantly increased luciferase activity in HEK293 cells comparedwith vehicle treated cells. The positive control LiCl also significantlyincreased luciferase activity (FIG. 12)

512 induces lung organoid growth in ex vivo cultures: Ex vivo 3Dorganoid cultures are one of the best models for studying stem cellgrowth and proliferation because organoid growth depends primarily onthe presence of stem cells. For these experiments, mice were euthanizedby CO2 or ketamine-xylazine administration and the abdominal aorta wasexposed and exsanguinated. The thoracic cavity was opened, and the lungswere exposed. Blood was flushed from the lung vasculature by perfusionwith 10 mL of sterile cold PBS through the right ventricle. The tracheawas cannulated with a 24-G cannula, and 1.2 mL of 10 U dispase (BD) wasthen injected into the lungs. The trachea and lungs were removed fromthe chest en-block and incubated for 20 minutes at room temperature(RT). The lung lobes were dissected from the trachea, heart, and rest ofmediastinal structures and then finely minced and incubated for 10 moremin with 2 mL of dispase. The suspension was passed through an 18-Gneedle 4-5 times to help open the lung compartments. If sticky DNA wasdetected, 10-30 μL of 4 mg/mL DNase I (Sigma) was added to the cellsuspension and incubated at 37° C. for 5 min. The cells were filteredthrough a 100-μ cell strainer (BD Biosciences) to obtain single-cellsuspensions (SCS). Red blood cells were lysed using RBC lysing buffer.Lung epithelial cells were resuspended in lung 3D culture media andmixed 2:1 with Cultrex Reduced Growth Factor Basement Membrane Extract,Type 2 (BD Biosciences). Then, 150 μL were placed into 24-well platesand incubated at 37° C. incubator for 20-30 min to solidify the matrix.A total of 600 μL lung 3D culture media was added carefully to the sideof the wells and kept in a 37° C. incubator. The media were changed 2-3times per week

Radiation exposure (6 Gy) inhibited organoid growth. However, treatmentwith BCN-512 (28 μM) at 1-hour post-radiation exposure mitigated theradiation damage and induced lung organoid growth (FIG. 13)

Pulmonary epithelial cells co-express Lgr5 and Fzd5 receptors inprogenitor cells located at the bronchoalveolar duct junction (BADJ):The Lgr5 receptor is associated with the Frizzled/Lrp Wnt receptorcomplex. R-Spondin1 is an intestinal mitogenic factor that binds to theLgr5 receptor and activates Wnt-β catenin signaling Lgr5 and Wntreceptor expression in the mouse lungs was assessed by performingimmunofluorescence staining of the mouse lung epithelium. Confocalmicroscopic images of mice lung epithelium clearly demonstrated thepresence of Lgr5- and Fzd5-positive cells (FIG. 14). It was noted thatthe Lgr5 and Fzd5 receptors were co-expressed in the BADJ region, whichis enriched in progenitor cells. However, most Lgr5- and Fzd5-positivecells disappeared within 5-7 days of irradiation with 18 Gy whole-thoraxlung irradiation (WTLI) (FIG. 14).

Example 10: Modulation of Macrophage Function

Myeloid cells were used to study the mechanism of action of theradiomitigator BCN-512 based on the following previous observations:

-   -   1. Lung irradiation activates macrophages both short- and        long-term (up to 6 months)    -   2. BCN-512 greatly increases the number of immature myeloid        cells that appear after whole body irradiation (WBI) and WTLI.        They co-express CD11b, Ly6G, and Ly6C, and are required for        mitigation by BCN-512, at least after WBI.    -   3. The mitigation of radiation lung damage by BCN-512 after LTI        is accompanied by a decrease in inflammatory macrophage content        and phenotypic markers in the lung on day 150.    -   4. 512 decreases the amount of pro-inflammatory cytokines        released by inflammatory peritoneal macrophages in response to        stimulation with LPS in vitro, suggesting that macrophage        function is modulated.    -   5. The mitigation of radiation lung damage by BCN-512 is        associated with an unexpected increase in anti-tumor activity;        we suspect that this also will be mediated by functional changes        in the macrophage population.

Because of the focus on lung fibrosis, we examined the effects ofirradiation on macrophages in terms of their ability to modulatefibroblast responses. We then used the most accepted and studied modelof inflammatory macrophages (stimulated peritoneal exudate cells), whichis also the model we used to show that BCN-512 affected pro-inflammatorycytokine production. The following in vitro experiments demonstrate thatBCN-512 can affect the function of macrophages by decreasing theactivation status and cytotoxicity of LPS-treated macrophages. Inaddition, BCN-512 blocks the ability of irradiated macrophages tostimulate fibroblast proliferation. The ability of BCN-512 to reprogrammacrophages is likely highly relevant to its ability to mitigateradiation induced fibrosis and tissue damage

Cell proliferation was used as a sensitive in vitro endpoint because itcan also be used to detect cell death. Cells were labeled with thefluorescent dye CFSE, which becomes diluted as the cells proliferateover time. We performed a series of experiments using tumor cell linesto validate the assay and determine the effective ratios of macrophagesto target cells (not shown) before performing experiments with normalearly-pass murine embryo fibroblasts. To study the effects of BCN-512and macrophages on fibroblast cell proliferation and death, irradiated(2 or 6 Gy) macrophages and fibroblasts were added at various ratios.Normal mouse fibroblasts were labelled with CFSE and added to themacrophages in 96-well plates. BCN-512 (10 μM) and/or LPS (1 μg/mL) ordiluent were then added, and the plates cultured for 1, 2, or 3 days.The purpose of the LPS was to activate the macrophages. BCN-512 wasadded 1 hour after LPS, which was added immediately after irradiation

The data showed that the fibroblasts divided every 15 hours. Low ratiosof non-irradiated macrophages at low ratios had little or no effect onfibroblast proliferation at any time point. However, at ratios of 20:1or 25:1, treating macrophages with LPS made them cytotoxic tofibroblasts (FIG. 19). This could be attributed to growth arrest andfibroblast cell-killing by activated macrophages, since LPS alone had nodirect effect on fibroblast proliferation or viability. There was nodifference between the groups of irradiated and non-irradiatedmacrophages, but BCN-512 practically abolished the toxicity associatedwith LPS treatment.

These findings are consistent with the concept that BCN-512 modulatesmacrophage function to suppress their inflammatory action; this requiresat least 2 days of co-culture, as shown in FIG. 16. FIG. 16 also showsthe tendency for normal macrophages to enhance the rate of fibroblastproliferation in vitro. In addition, 2 Gy irradiation enhanced thissupportive action, although the results were not statisticallysignificant. After 6 Gy macrophage irradiation (FIG. 17), the number ofviable fibroblasts clearly increased by 30-50% in all cases by 3 days;the addition of BCN-512 seemed to neutralize this function

FIG. 18 shows that local thoracic radiation (14.5Gy) of mice induceslung damage in the absence of BCN057. A: Lung histology of C57BL/6 micereceiving local thoracic radiation (radiation of the lung area)presenting a focal area of increased cellularity and edema on the upperleft quadrant. Adjacent to this is increased cellularity (the laceypatterning). The lower right quadrant, emphysema is present with largeclear areas indicating where alveoli have collapsed to present largeopen areas. B. Histopathology of C57BL/6 mouse lung under identicalradiation treatment as A, but also treated with 5 mg/kg BCN512 once perday every 24 hours for 5 doses. Lung tissue is normal with no evidentemphysema or edema or hyper cellularity or immune infiltrate.

FIG. 19 shows lung fibrosis: A, B are differing whole lobe sections fromthe same animal lung having received 14.5Gy local thoracic radiation(Day 120 after irradiation treatment). In both lobes, significantcollapse of alveoli can be seen along with hyper cellularity (darkareas) and pronounced emphysema (large open areas) presenting fibrosis.C, D are separate lobes from the same animal treated identically asabove but receiving BCN512 at 5 mg/kg daily for 5 days after 14.5Gylocal thoracic radiation. Absent are the large lesions and pronouncedfibrosis.

In summary for the above data, these in vitro experiments demonstratedthat BCN-512 can affect the function of macrophages. It decreased theactivation status and cytotoxicity of LPS-treated macrophages. Fornon-LPS treated macrophages, BCN-512 blocked the ability of irradiatedmacrophages to stimulate fibroblast proliferation. Fibroblastproliferation is macrophage dependent and 512 inhibits this macrophagefunction. The ability of BCN-512 to reprogram macrophages is likelyhighly relevant to its ability to mitigate radiation damage

The absence of late effects in fibrosis from animals from long termradiation (observing from long term studies of the total bodyirradiation studies) along with the reduction of fibroblasts indicatethese drugs are effective at preventing fibrosis and inflammation fromboth radiation or chemical means (LPS). Organoid structures are grownfrom stem cells to differentiate into the features of the organ theycame from. The drugs activate wnt signaling of which, stem cells are acritical target population of cells that respond to wnt by self-renewaland differentiation. Stem cells are important for the repair andregeneration of tissues that are damaged and therefore these drugspreserve stem cells to allow for normal tissue repair. In the normalcase, these stem cells are destroyed by radiation or chemotherapy forexample which allows inflammatory macrophages and immune infiltrates tocome in and remodel the tissue with consequent fibrosis in the latestage.

Example 11: Dermal Wound Healing

FIG. 20 shows the sites of radiation described in Table 2.

TABLE 2 Average Draize scoring for treatment of control, (no radiation),BCN512 or BCN057 at day 15 post dermal radiation receiving radiationdoses of 25, 35 and 45 Gy according to table Y below. The day ofirradiation will is Day 0. Animals receive localized irradiation on Day0. A total of six sites, measuring approximately 4 × 4 cm, areirradiated: two sites on the dorsolateral aspect of the neck (left andright), two sites on each hind limb (lateral and medial aspect of thethigh). Dermal dosing formulations are applied to the dermal treatmentsites on Day 0 following irradiation. The following applications (Day 1to 6) were performed on the same time of the day of the firstapplication. Day Erythema Erythema Erythema Erythema Erythema EdemaEdema Edema Edema Edema # Site 2 Site 3 Site 4 Site 5 Site 6 Edema Site2 Site 3 Site 4 Site 5 Site 6 Ctrl 1 0 1 0 1 1 0 0 1 0 1 0 2 0 1 0 1 0 00 1 0 1 0 3 0 1 0 1 1 0 0 1 0 1 0 4 0 2 0 1 1 0 0 0 0 0 0 5 1 2 0 2 0 00 1 0 1 0 6 1 2 1 2 1 0 0 1 0 1 0 8 1 2 1 2 1 0 0 0 0 0 0 0.428 1.5710.285 1.428 0.714 0.714 0.714 Avg 571 429 714 571 286 0 0 286 0 286 0BCN512 1 0 1 1 2 1 0 0 0 0 1 0 2 1 1 0 1 1 0 0 1 0 1 0 3 1 1 0 1 1 0 0 10 1 0 4 0 1 0 1 0 0 0 0 0 0 0 5 1 2 0 2 1 0 0 1 0 1 0 6 0 2 0 2 1 0 0 10 1 0 8 0 2 0 2 1 0 0 0 0 1 0 0.428 1.428 0.142 1.571 0.857 0.571 0.857Avg 571 571 857 429 143 0 0 429 0 143 0 BCN057 1 0 1 0 1 1 0 0 1 0 0 0 20 1 0 1 1 0 0 1 0 0 0 3 1 1 0 1 0 0 0 1 0 1 0 4 0 1 0 1 0 0 0 0 0 0 0 50 1 0 2 0 0 0 0 0 1 0 6 0 2 1 2 1 0 0 1 0 1 0 8 0 2 0 2 0 0 0 0 0 0 00.142 1.285 0.142 1.428 0.428 0.571 0.428 Avg 857 714 857 571 571 0 0429 0 571 0

TABLE 3 Legend for draize scoring ERYTHEMA/ESCHAR FORMATION (MaximumScore = 4) SCORE No erythema 0 Very slight erythema, barely perceptible(edges are not defined) 1 Well-defined erythema (pale red in color) 2Moderate to severe erythema (definite red in color) 3 Severe erythema(beet or crimson red in color) and/or eschar 4 formation (scabformation) EDEMA FORMATION (Maximum score = 4) SCORE No edema 0 Veryslight edema, barely perceptile (edges are not defined) 1 Slight edema(edges are not definable but the area is slightly 2 raised) Moderateedema (area well-defined and raised approximately 3 mm) Severe edema(raised more than 1 mm and extending beyond the 4 area of exposure)

TABLE 4 Study Design (radiation treatment) Radiation Dose Level (Gy)Group Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Animals 1 - Control 2535 45 1 2 Group 2 - 1 BCN512 3 Group 3 - 1 BCN057

TABLE 5 Study Design (dermal treatment) Dose Dose Conc. Volume/ Group(mg/mL) Site Animals 1 Control* 0 0.5 ml 1 2 Group 2- 10 mg/mL 1 BCN5123 Group 3- 10 mg/mL 1 BCN057 *Group 1 animal receive the referenceitem/vehicle, Dimethyl sulfoxide, DMSO in sites 1, 3 and 5 only, sites2, 4 and 6 sites will not receive the reference item/vehicle.

FIG. 24 shows photographs of the wounds analyzed with Draize scoring.The radiation induced dermatitis study show that the drugs reduceradiation dermatitis. This condition is a dose limiting condition ofradiation therapy where the skin is subject to radiation and responds inan inflammatory state presenting erythema and edema. LGR5+ stem cellsare present in dermis and are equally affected by radiation. Because ofthe effects of the drugs on inflammation and stem cell preservation,these drugs protect against radiation dermatitis and support tissueregeneration in the case of injury.

Example 12: BCN057 pH Effect on Formulation

TABLE 6 BCN057 formulation. Species 1-4 are shown in FIG. 22. The pH ofBCN057 is required to be below 5 in order to take a proton and be watersoluble. We have successfully used cylodextrins with BCN057 which willwork if you drive it into solution at low pH. It will not work if you donot solubilize it first. In conclusion, the reduction in pH below 4.0provides a soluble version of BCN057 and this gives significantadvantage in drug handling and formulation. pH 1% 2% 3% 4% Species 0 099.85 0 0.1 0.2 0 99.9 0 0.07 0.4 0 99.94 0 0.04 0.6 0 99.96 0 0.03 0.80.01 99.97 0 0.02 1 0.01 99.98 0 0.01 1.2 0.01 99.98 0 0.01 1 1.4 0.0299.97 0 0 1.6 0.04 99.96 0 0 1.8 0.06 99.94 0 0 2 0.09 99.9 0 0 2.2 0.1599.85 0 0 2.4 0.23 99.76 0 0 2.6 0.37 99.63 0 0 2.8 0.59 99.41 0 0 2 30.93 99.07 0 0 3.2 1.46 98.54 0 0 3.4 2.3 97.7 0 0 3.6 3.59 96.41 0 03.8 5.58 94.42 0 0 4 8.56 91.44 0 0 4.2 12.92 87.08 0 0 4.4 19.04 80.960 0 3 4.6 27.16 72.84 0 0 4.8 37.14 62.86 0 0 5 48.36 51.64 0 0 5.259.75 40.25 0 0 5.4 70.17 29.83 0 0 5.6 78.85 21.15 0 0 5.8 85.53 14.470 0 6 90.35 9.65 0 0 4 6.2 93.69 6.31 0 0 6.4 95.92 4.08 0 0 6.6 97.392.61 0 0 6.8 98.34 1.66 0 0 7 98.94 1.06 0 0 7.2 99.33 0.67 0 0 7.499.58 0.42 0 0 7.6 99.73 0.27 0 0 7.8 99.83 0.17 0 0 8 99.89 0.11 0 08.2 99.93 0.07 0 0 8.4 99.95 0.04 0 0 8.6 99.97 0.03 0.01 0 8.8 99.970.02 0.01 0 9 99.97 0.01 0.02 0 9.2 99.97 0.01 0.03 0 9.4 99.95 0 0.04 09.6 99.93 0 0.07 0 9.8 99.89 0 0.11 0 10 99.83 0 0.17 0 10.2 99.73 00.27 0 10.4 99.58 0 0.42 0 10.6 99.33 0 0.67 0 10.8 98.94 0 1.06 0 1198.34 0 1.66 0 11.2 97.39 0 2.61 0 11.4 95.92 0 4.08 0 11.6 93.69 0 6.310 11.8 90.35 0 9.65 0 12 85.53 0 14.47 0 12.2 78.86 0 21.14 0 12.4 70.180 29.82 0 12.6 59.75 0 40.25 0 12.8 48.37 0 51.63 0 13 37.15 0 62.85 013.2 27.16 0 72.84 0 13.4 19.05 0 80.95 0 13.6 12.93 0 87.07 0 13.8 8.570 91.43 0 14 5.58 0 94.42 0

REFERENCES

-   1. Monks, A.; Scudiero, D. A.; Skehan, P.; Shoemaker, R. H.;    Paull, K. D.; Vistica, D. T.; Hose, C.; Langley, J.; Cronice, P.;    Vaigro-Wolf, M.; Gray-Goodrich, M.; Campbell, H.; Mayo, M. R.    JNCI, J. Natl. Cancer Inst. 1991, 83, 757-766.-   2. Alley, M. C.; Scudiero, D. A.; Monks A.; Hursey, M. L.;    Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, A.;    Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589-601.-   3. Shoemaker, R. H.; Monks, A.; Alley, M. C.; Scudiero, D. A.;    Fine, D. L.; McLemore, T. L.; Abbott, B. J.; Paull, K. D.; Mayo, J.    G.; Boyd, M. R. Prog. Clin. Biol. Res. 1988, 276, 265-286.-   4. Stinson, S. F.; Alley, M. C.; Kenny, S.; Fiebig, H.; Boyd, M. R.    Proc. Am. Assoc. Cancer Res. 1989, 30,613.-   5. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;    Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R.    JNCI, J. Natl. Cancer Inst. 1990, 82, 1113-1118.-   6. Skehan, P.; Storeng, R.; Scudiero, D. A.; Monks, A.; McMahon, J.;    Vistica, D. T.; Warren, J. T.; Bokesch, H.; Kenny, F.; Boyd, M. R.    JNCI, J. Natl. Cance

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A method of increasing hematopoiesis in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of BCN057 or an analog thereof.


2. The method of claim 1, wherein the subject has one or more of bloodcancer, lymphoma, leukemia, AML, ALL, bone marrow ablation, bone marrowtransplant, bone marrow suppression, a platelet disorder,thrombocytopenia, Von Willebrand disease, and clinical radiation andchemotherapy related exposure.
 3. The method of claim 2, wherein theplatelet disorder is caused by one or more of bone marrow failure, bonemarrow suppression, chronic alcohol abuse, congenitalmacrothrombocytopenias, viral infection, bacterial infection,myelodysplastic syndrome, neoplastic marrow infiltration, a nutritionaldeficiency, radiation and chemotherapy.
 4. The method of claim 1,wherein the analog of BCN057 is selected from the group consisting ofFormula IB, Formula IC, Formula ID, Formula IE, Formula IF, Formula IGand Formula IH:


5. The method of claim 1, wherein the subject received radiation therapyand/or chemotherapy.
 6. The method of claim 3, wherein the bone marrowfailure is caused by one or more of aplastic anemia, paroxysmalnocturnal hemoglobinuria and Shwachman-Diamond syndrome.
 7. The methodof claim 3, wherein the bone marrow suppression is caused by one or moreof medication, chemotherapy and irradiation.
 8. The method of claim 3,wherein the congenital macrothrombocytopenia is one or more of Alportsyndrome, Bernard-Soulier syndrome, Fanconi anemia, platelet-type orpseudo-von Willebrand disease and Wiskott-Aldrich syndrome.
 9. Themethod of claim 3, wherein the viral infection is one or more ofcytomegalovirus, Epstein-Barr virus, hepatitis C virus, HIV, mumps,parvovirus B19, rickettsia, rubella and varicella-zoster virus.
 10. Themethod of claim 3, wherein the nutritional deficiency is a lack of oneor more of vitamin B12 and folate.